Cellular arrays and methods of detecting and using genetic disorder markers

ABSTRACT

A method is disclosed for rapid molecular profiling of tissue or other cellular specimens by placing a donor specimen in an assigned location in a recipient array, providing copies of the array, and performing a different biological analysis of each copy. The results of the different biological analyses are compared to determine if there are correlations between the results of the different biological analyses at each assigned location. In some embodiments, the specimens may be tissue specimens from different tumors, which are subjected to multiple parallel molecular (including genetic and immunological) analyses. The results of the parallel analyses are then used to detect common molecular characteristics of the genetic disorder type, which can subsequently be used in the diagnosis or treatment of the disease. The biological characteristics of the tissue can be correlated with clinical or other information, to detect characteristics associated with the tissue, such as susceptibility or resistance to particular types of drug treatment. Other examples of suitable tissues which can be placed in the matrix include tissue from transgenic or model organisms, or cellular suspensions (such as cytological preparations or specimens of liquid malignancies or cell lines).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/106,038, filed on Oct. 28, 1998, and U.S. Provisional ApplicationSer. No. 60/150,493, filed on Aug. 24, 1999, both of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the screening of tissue samples and genomicregions to discover markers for genetic disorders such as cancer.

BACKGROUND OF THE INVENTION

Biological mechanisms of many diseases have been clarified bymicroscopic examination of tissue specimens. Histopathologicalexamination has also permitted the development of effective medicaltreatments for a variety of illnesses. In standard anatomical pathology,a diagnosis is made on the basis of cell morphology and stainingcharacteristics. Tumor specimens, for example, can be examined tocharacterize the tumor type and predict the aggressiveness of the tumor.Although this microscopic examination and classification of tumors hasimproved medical treatment, the microscopic appearance of a tissuespecimen stained by standard methods (such as hematoxylin and eosin) canoften reveal only a limited amount of diagnostic or molecularinformation.

Recent advances in molecular medicine have provided an even greateropportunity to understand the cellular mechanisms of disease, and selectappropriate treatments with the greatest likelihood of success. Somehormone dependent breast tumor cells, for example, have an increasedexpression of estrogen receptors on their cell surfaces, which indicatesthat the patient from whom the tumor was taken will likely respond tocertain anti-estrogenic drug treatments. Other diagnostic and prognosticcellular changes include the presence of tumor specific cell surfaceantigens (as in melanoma), the production of embryonic proteins (such asα-fetoprotein in liver cancer and carcinoembryonic glycoprotein antigenproduced by gastrointestinal tumors), and genetic abnormalities (such asactivated oncogenes in tumors). A variety of techniques have evolved todetect the presence of these cellular abnormalities, includingimmunophenotyping with monoclonal antibodies, in situ hybridization withprobes, and DNA amplification using the polymerase chain reaction (PCR).

The development of new molecular markers of clinical importance has beenimpeded by the slow and tedious process of evaluating biomarkers inlarge numbers of clinical specimens. For example, hundreds of tissuespecimens representing different stages of tumor progression have to beevaluated before the importance of a given marker can be assessed. Sincethe number of antibodies, as well as probes for mRNA or DNA targets isincreasing rapidly, only a small fraction of these can ever be tested inlarge numbers of clinical specimens.

Various methods have been explored to prepare samples of multipletissues or nucleic acids on one slide or plate.

SUMMARY OF THE INVENTION

The invention is based on the discovery that two very different types ofarrays can be used in combination in new methods to rapidly andaccurately detect, with high resolution, genomic copy numberalterations, such as gene amplifications or deletions, that can serve asmarkers for various genetic disorders such as cancers and trisomies.

Thus, the invention features methods of detecting particular genomic“target” regions (nucleic acid sequences) that correspond to specificgenetic disorders, e.g., one or more different types of tumors orhereditary genetic diseases, by combining tissue microarray technologywith other technologies, such as high-throughput genomics. These methodsare used to identify molecular characteristics, such as structuralchanges in genes or proteins, and copy number or expression alterationsof genes, and to correlate these results with disease prognosis ortherapy outcome to identify novel targets for gene prevention, earlydiagnosis, disease classification, or prognosis, and to identifytherapeutic agents. High-throughput technologies include cDNA arrays andComparative Genomic Hybridization (“CGH”) arrays.

The invention also includes methods of preparing new arrays of nucleicacids (genes), e.g., representative of specific types of tumors(tumor-specific diagnostic gene arrays); probes that hybridizeselectively to these genomic target regions; methods of preparing theprobes; methods of using the probes to screen for and/or diagnosespecific genetic disorders; compositions that interact with the genomictarget regions to treat the genetic disorders; and methods of treatingthe genetic disorders using these compositions.

In general, in one aspect the invention features a method of parallelanalysis of tissue specimens, by obtaining a plurality of donorspecimens; placing each donor specimen in an assigned location in arecipient array; using a genosensor comparative genomic hybridization(gCGH) array to identify a biomarker to test on the recipient array;obtaining a plurality of sections from the recipient array in a mannerthat each section contains a plurality of donor specimens that maintaintheir assigned locations; performing on each section a differentbiological analysis using the biomarker; and comparing the results ofthe different biological analyses in corresponding assigned locations ofdifferent sections to determine if there are correlations between theresults of the different biological analyses at each assigned location.For example, the biomarker can be selected by high-throughput geneticanalysis, and the biomarker can include a numerical alteration of achromosome, chromosomal region, gene, gene fragment, or locus.

The results can be compared by determining if there is an alteration ofa gene by examining a marker for gene alteration. For example, thealteration can be an amplification of PDGFB in breast, lung, colon,testicular, endometrial, or bladder cancer.

In another embodiment, the invention features a method of analyzing geneamplification in a tissue specimen by screening multiple genes in atissue specimen with a genosensor comparative genomic hybridization(gCGH) array that detects which genes are amplified in the tissuespecimen; and screening multiple tissue specimens in a tissue array witha nucleic acid probe to detect which genes are amplified in the tissuespecimens; wherein the result of screening multiple genes is used toselect the nucleic acid probe to screen the multiple tissue specimens,or wherein the result of screening multiple tissue specimens is used toselect the array that detects which genes are amplified.

In this method, the gCGH array can be assayed for a gene amplification,or a genetic or molecular marker that reflects this amplification. TheCGH array can be a microarray that contains target loci that undergoamplification in cancer.

The invention also features a method of analyzing a biological samplefor a genetic disorder by exposing a genosensor comparative genomichybridization (gCGH) array of genomic regions to a nucleic acid samplefrom a cell with a known specific genetic disorder, and identifying as abiomarker a genomic region to which the nucleic acid hybridizes;obtaining a candidate probe that hybridizes to the biomarker; exposingthe candidate probe to a tissue specimen array to determine astatistical measure of hybridization of the candidate probe; selecting acandidate probe having a statistically significant measure ofhybridization; and using a selected candidate probe to analyze abiological sample for the genetic disorder. This analysis of thebiological sample can provide diagnostic or prognostic information.

In addition, the invention features a method of detecting the presenceof cancerous cells in a specimen, by determining whether plateletderived growth factor beta (PDGFB) is amplified in the specimen,amplification indicating the presence of cancerous cells in thespecimen, e.g., a lung, bladder, or endometrial tissue specimen.

In another aspect, the invention features a method for detecting agenomic target sequence that is associated with a specific geneticdisorder by contacting a plurality of genomic regions in a genosensorcomparative genomic hybridization (gCGH) array with a nucleic acid testsample including nucleic acid fragments that collectively represent DNAfrom a cell with a known specific genetic disorder under conditions thatallow the nucleic acid fragments to hybridize to one or more candidategenomic regions; measuring the amount of nucleic acid test samplehybridized to the candidate genomic regions, if any, and selecting acandidate genomic region corresponding to an altered amount ofhybridized test sample nucleic acid compared to a control sample ofnormal DNA; preparing a nucleic acid probe that hybridizes to theselected candidate genomic region; contacting a plurality of tissuesamples with the probe under conditions that allow the probe tohybridize to nucleotide sequences in the tissue samples; and selecting acandidate genomic region corresponding to a probe that hybridizes to asignificant number of tissue samples as a genomic target sequence thatis associated with the specific genetic disorder.

As used herein, a “polypeptide” is any chain of amino acids, regardlessof length or post-translational modification (e.g., glycosylation orphosphorylation).

A “gene amplification” is an increase in the copy number of a gene, ascompared to the copy number in normal tissue. An example of a genomicamplification is an increase in the copy number of an oncogene. A “genedeletion” is a deletion of one or more nucleic acids normally present ina gene sequence, and in extreme examples can include deletions of entiregenes or even portions of chromosomes.

A “genomic target sequence” is a sequence of nucleotides located in aparticular region in the human genome that corresponds to one or morespecific loci, including genetic abnormalities, such as a nucleotidepolymorphism, a deletion, or an amplification.

A “genetic disorder” is any illness, disease, or abnormal physical ormental condition that is caused by an alteration in one or more genes orregulatory sequences (such as an amplification, mutation, deletion, ortranslocation).

“Comparative Genomic Hybridization” or CGH is a technique ofdifferential labeling of test DNA and normal reference DNA, which arehybridized simultaneously to chromosome spreads, as described inKallioniemi et al., Science, 258:818-821, 1992.

A “nucleic acid array” refers to an arrangement of nucleic acid (such asDNA or RNA) in assigned locations on a matrix, such as that found incDNA or CGH arrays.

A “microarray” is an array that is miniaturized so as to requiremicroscopic examination for visual evaluation.

A “DNA chip” is a DNA array in which multiple DNA molecules (such ascDNAs) of known DNA sequences are arrayed on a substrate, usually in amicroarray, so that the DNA molecules can hybridize with nucleic acids(such as cDNA or RNA) from a specimen of interest. DNA chips are furtherdescribed in Ramsay, Nature Biotechnology, 16:40-44, 1998.

“Gene expression microarrays” refers to microscopic arrays of cDNAsprinted on a substrate, which serve as a high density hybridizationtarget for mRNA probes, as in Schena, BioEssays 18:427-431, 1996.

“Serial Analysis of Gene Expression” or “SAGE” refers to the use ofshort sequence tags to allow the quantitative and simultaneous analysisof a large number of transcripts in tissue, as described in Velculescuet al., Science, 270:484-487, 1995.

“High throughput genomics” refers to application of genomic or geneticdata or analysis techniques that use microarrays or other genomictechnologies to rapidly identify large numbers of genes or proteins, ordistinguish their structure, expression or function from normal orabnormal cells or tissues.

A “tumor” is a neoplasm that may be either malignant or non-malignant.“Tumors of the same tissue type” refers to primary tumors originating ina particular organ (such as breast, prostate, bladder or lung). Tumorsof the same tissue type may be divided into tumors of differentsub-types (a classic example being bronchogenic carcinomas (lung tumors)which can be an adenocarcinoma, small cell, squamous cell, or large celltumor).

A “cellular” specimen is one which contains whole cells, and includestissues, which are aggregations of similarly specialized cells united inthe performance of a particular function. Examples include cells fromthe skin, breast, prostate, blood, testis, ovary and endometrium.

A “cellular suspension” is a liquid in which cells are dispersed, andmay include a uniform or non-uniform suspension. Examples of cellularsuspensions are those obtained by fine-needle aspiration from tumorsites, cytology specimens (such as vaginal fluids for preparing Papsmears, washes (such as bronchial washings), urine that contains cells(for example in the detection of bladder cancer), ascitic fluid (forexample obtained by abdominal paracentesis), or other body fluids.

A “cytological preparation” is a pathological specimen, such as vaginalfluids, in which a cellular suspension can be converted into a smear orother form for pathological examination or analysis.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The invention provides a rapid means of identifying not only specificgenomic abnormalities present in a tissue, but the importance andstatistical significance of these abnormalities in hundreds or thousandsof tissues, to provide relevant diagnostic and prognostic information,as well as potential targets for therapeutic agents.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic perspective view of a first embodiment of thepunch device of the present invention, showing alignment of the punchabove a region of interest of donor tissue in a donor block.

FIG. 2 is a view similar to FIG. 1, but in which the punch has beenadvanced to obtain a donor specimen sample.

FIG. 3 is a schematic, perspective view of a recipient block into whichthe donor specimen has been placed.

FIGS. 4-8 are schematic diagrams illustrating steps in the preparationof thin section arrays from the recipient block.

FIG. 9 is a perspective view of a locking device for holding a slidemounted specimen above the tissue in the donor block to locate a regionof interest.

FIG. 10A is a view of an H&E stained, thin section tissue array mountedon a slide for microscopic examination.

FIG. 10B is a magnified view of a portion of the slide in FIG. 10A,showing results of erbB2 mRNA in situ hybridization on a tissue arrayfrom the region in the small rectangle in FIG. 10A.

FIG. 10C is an electrophoresis gel showing that high molecular weightDNA and RNA can be extracted from the breast cancer specimens fixed incold ethanol.

FIG. 10D is an enlarged view of one of the tissue samples of the arrayin FIG. 10A, showing an immunoperoxidase staining for the erbB2 antigen.

FIG. 10E is a view similar to FIG. 10D, showing high level erbB2 geneamplification detected by fluorescent in situ hybridization (FISH) oftissue in the array by an erbB2 DNA probe.

FIGS. 11A, 11B, 11C and 11D are schematic views illustrating an exampleof parallel analysis of arrays obtained by the method of the presentinvention.

FIG. 12 is an enlarged view of a portion of FIG. 11.

FIG. 13 is a schematic representation of a genosensor CGH microarraythat contains 31 target loci that have been reported to undergoamplification in cancer. Circles around target loci indicateamplifications found in the breast cancer cell lines tested in thisstudy.

FIG. 14 is a digital representation of the results of a chromosomal CGHanalysis showing high level amplifications in Sum-52 breast cancer cellsat 10q25-q26 and at 7q21-q22, a genosensor CGH analysis indicating highlevel amplifications of the MET (7q21) and FGFR2 (10q25) oncogenes, anda FISH analysis showing amplification of FGFR2 (at 10q25).

FIG. 15 is a schematic diagram of a breast cancer tissue microarray, aswell as a digital image of a hybridization, showing that FGFR2 wasamplified in 4.5% of the tumor samples in the breast cancer tissuemicroarray.

FIG. 16 is a schematic representation of the combination of the DNAarray and the tissue array, showing that the DNA array can probe asingle tumor with hundreds of probes, while the tissue array technologycan conversely probe specimens from hundreds of tumors with a singleprobe.

FIG. 17 is a schematic diagram representing the combination of thetissue array technology with cDNA and/or CGH arrays.

DETAILED DESCRIPTION

The use of tissue arrays in combination with other array techniques canprovide information about the frequency of a multitude of geneticalteration or gene expression patterns (including normal gene expressionpatterns) in a variety of tissue types (such as different types oftumors), and in tissue of a particular histological type (such as atumor of a specific type, such as intraductal breast cancer), as well asthe tissue distribution of molecular markers tested.

In one specific embodiment of the combined DNA and tissue arrays, theDNA array may be a cDNA or genomic microarray chip that allows aplurality (hundreds, thousands, or even more) of different nucleic acidsequences to be affixed to the surface of a support to form an array.Such a chip may, for instance carry an array of cDNA clones,oligonucleotides, or large-insert genomic P1, BAC, or PAC clones. Thesearrays enable the analysis of hundreds of genes or genomic fragments atonce to determine their expression or copy number in a test specimen.

A high-throughput DNA chip can be used together with high-throughputtissue array technology. Such hybrid inventions include using a DNAarray to screen a limited number of tumor samples for expression or copynumber of one or more (for example thousands of) specific genes or DNAsequences. Probes containing the gene of interest may then be used toscreen a tissue microarray that contains many different tissue specimens(such as a variety of breast tumors or prostate tumors) to determine ifthe identified gene or genetic locus is similarly altered in thesetumors. For instance, a cDNA chip can be used to screen a human breastcancer cell line, to identify one or more genes that are overexpressedor amplified in that particular breast cancer. A probe, corresponding tothe identified gene, would then be used to probe a tissue arraycontaining a plurality of tissue samples from different breast cancers,or even tumors of different types (such as lung or prostate cancer).Such a probe could be made by labeling the identical clone used in theDNA array (for example with a fluorescent or radioactive marker). Thepresence of the gene in related (or unrelated) tumors would be revealedby the pattern of hybridization of the probe to the tissue array.

Another embodiment includes a method of preparing a diagnostictumor-specific gene array.

Embodiments of FIGS. 1 to 12

A first embodiment of a device for making the microarrays of the presentinvention is shown in FIGS. 1 and 2, in which a donor block 30 (oftissue) is shown in a rectangular container 31 mounted on a stationaryplatform 32 having an L-shaped edge guide 34 that maintains donorcontainer 31 in a predetermined orientation on platform 32. A punchapparatus 38 is mounted above platform 32, and includes a vertical guideplate 40 and a horizontal positioning plate 42. The positioning plate 42is mounted on an x-y stage (not shown) that can be precisely positionedwith a pair of digital micrometers.

Vertical guide plate 40 has a flat front face that provides a precisionguide surface against which a reciprocal punch base 44 can slide along atrack 46 between a retracted position shown in FIG. 1 and an extendedposition shown in FIG. 2. An elastic band 48 helps control the movementof base 44 along this path, and the limits of advancement and retractionof base 44 are set by track member 46, which forms a stop that limitsthe amplitude of oscillation of base 44. A thin wall stainless steeltube punch 50 with sharpened leading edges is mounted on the flat bottomface of base 44, so that punch 50 can be advanced and retracted withrespect to platform 32, and the container 31 on the platform. The hollowinterior of punch 50 is continuous with a cylindrical bore through base44, and the bore opens at opening 51 on a horizontal lip 53 of base 44.

FIG. 1 shows that a thin section of tissue, stained withhematoxylin-eosin or other stains, can be obtained from donor block 30and mounted on a slide 52 (with appropriate preparation and staining) sothat anatomic and micro-anatomic structures of interest can be locatedin the block 30. Slide 52 can be held above donor block 30 by anarticulated arm holder 54 (FIG. 9) with a clamp 56 which securely holdsan edge of a transparent support slide 58. Arm holder 54 can articulateat joint 60, to swivel between a first position in which support slide58 is locked in position above container 31, and a second position inwhich support slide 58 moves horizontally out of the position shown inFIG. 9 to permit free access to punch 50.

In operation, the rectangular container 31 is placed on platform 32(FIG. 1) with edges of container 31 abutting edge guides 34 to holdcontainer 31 in a selected position. A donor block 30 is prepared byembedding a gross tissue specimen (such as a three dimensional tumorspecimen 62) in paraffin. A thin section of donor block 30 is shavedoff, stained, and mounted on slide 52 as thin section 64, and slide 52is then placed on support slide 58 and positioned above donor block 30as shown in FIG. 9. Slide 52 can be moved around on support slide 58until the edges of thin section 64 are aligned with the edges of thegross pathological specimen 62, as shown by the dotted lines in FIG. 9.Arm 54 is then locked in the first position, to which the arm cansubsequently return after displacement to a second position.

A micro-anatomic or histologic structure of interest 66 can then belocated by examining the thin section through a microscope (not shown).If the tissue specimen is, for example, an adenocarcinoma of the breast,then the location of the structure of interest 66 may be an area of thespecimen in which the cellular architecture is suggestive of specificfeatures of the cancer, such as invasive and noninvasive components.Once the structure of interest 66 is located, the corresponding regionof tissue specimen 62 from which the thin section structure of interest66 was obtained is located immediately below the structure of interest66. As shown in FIG. 1, positioning plate 42 can be moved in the x and ydirections (under the control of the digital micrometers or a joystick),or the donor block can be moved for larger distances, to align punch 50in position above the region of interest of the donor block 30, and thesupport slide 58 is then horizontally pivoted away from its positionabove donor block 30 around pivot joint 60 (FIG. 9).

Punch 50 is then introduced into the structure of interest in donorblock 30 (FIG. 2) by advancing vertical guide plate 40 along track 46until plate 44 reaches its stop position (which is preset by apparatus38). As punch 50 advances, its sharp leading edge bores a cylindricaltissue specimen out of the donor block 30, and the specimen is retainedwithin the punch as the punch reciprocates back to its retractedposition shown in FIG. 1. The cylindrical tissue specimen cansubsequently be dislodged from punch 50 by advancing a stylet (notshown) into opening 51. The tissue specimen is, for example, dislodgedfrom punch 50 and introduced into a cylindrical receptacle ofcomplementary shape and size in an array of receptacles in a recipientblock 70 shown in FIG. 3.

One or more recipient blocks 70 can be prepared prior to obtaining thetissue specimen from the donor block 30. Block 70 can be prepared byplacing a solid paraffin block in container 31 and using punch 50 tomake cylindrical punches in block 70 in a regular pattern that producesan array of cylindrical receptacles of the type shown in FIG. 3. Theregular array can be generated by positioning punch 50 at a startingpoint above block 70 (for example a corner of the prospective array),advancing and then retracting punch 50 to remove a cylindrical core froma specific coordinate on block 70, then dislodging the core from thepunch by introducing a stylet into opening 51. The punch apparatus orthe recipient block is then moved in regular increments in the x and/ory directions, to the next coordinate of the array, and the punching stepis repeated. In the specific disclosed embodiment of FIG. 3, thecylindrical receptacles of the array have diameters of about 0.6 mm,with the centers of the cylinders being spaced by a distance of about0.7 mm (so that there is a distance of about 0.05 mm between theadjacent edges of the receptacles).

In a specific example, core tissue biopsies having a diameter of 0.6 mmand a height of 3-4 mm, were taken from selected representative regionsof individual “donor” paraffin-embedded tumor blocks and preciselyarrayed into a new “recipient” paraffin block (20 mm×45 mm). H&E-stainedsections were positioned above the donor blocks and used to guidesampling from morphologically representative sites in the tumors.Although the diameter of the biopsy punch can be varied, 0.6 mmcylinders have been found to be suitable because they are large enoughto evaluate histological patterns in each element of the tumor array,yet are sufficiently small to cause only minimal damage to the originaldonor tissue blocks, and to isolate reasonably homogenous tissue blocks.

Up to 1000 such tissue cylinders, or more, can be placed in one 20×45 mmrecipient paraffin block. Specific disclosed diameters of the cylindersare 0.1-4.0 mm, for example 0.5-2.0 mm, and most specifically less than1 mm, for example 0.6 mm. Automation of the procedure, with computerguided placement of the specimens, allows very small specimens to beplaced tightly together in the recipient array.

FIG. 4 shows the array in the recipient block after the receptacles ofthe array have been filled with tissue specimen cylinders. The topsurface of the recipient block is then covered with an adhesive film 74from an adhesive coated tape sectioning system (Instrumedics) to helpmaintain the tissue cylinder sections in place in the array once it iscut. The array block may be warmed at 37° C. for 15 minutes beforesectioning, to promote adherence of the tissue cores and allow smoothingof the block surface when pressing a smooth, clean surface (such as amicroscope slide) against the block surface.

With the adhesive film in place, a 4-8 μm section of the recipient blockis cut transverse to the longitudinal axis of the tissue cylinders (FIG.5) to produce a thin microarray section 76 (containing tissue specimencylinder sections in the form of disks) that is transferred to aconventional specimen slide 78. The microarray section 76 is adhered toslide 78, for example by adhesive on the slide. The film 74 is thenpeeled away from the underlying microarray member 76 to expose it forprocessing. A darkened edge 80 of slide 78 is suitable for labeling orhandling the slide.

Breast cancer tissue specimens were fixed in cold ethanol to helppreserve high-molecular weight DNA and RNA, and 372 of the specimenswere fixed in this manner. At least 200 consecutive 4-8 μm tumor arraysections can be cut from each block providing targets for correlated insitu analyses of multiple molecular markers at the DNA, RNA, or proteinlevel, including copy number or expression of multiple genes. Thisanalysis is performed by testing for different gene molecular targets(e.g., DNA or RNA sequences or antigens defined by antibodies) inseparate array sections, and comparing the results of the tests atidentical coordinates of the array (which correspond to tissue specimensfrom the same tissue cylinder obtained from donor block). This approachenables measurement of virtually hundreds of molecular characteristicsfrom every tumor, thereby facilitating construction of a large series ofcorrelated genotypic or phenotypic characteristics of uncultured humantumors.

An example of a single microarray 76 containing 645 specimens is shownin FIG. 10A. An enlarged section of the microarray (highlighted by arectangle in FIG. 10A) is shown in FIG. 10B, in which an autoradiogramof erbB2 mRNA in situ hybridization illustrates that two adjacentspecimens in the array demonstrate a strong hybridization signal. FIG.10C illustrates electrophoresis gels which demonstrate that highmolecular weight DNA and RNA can be extracted from breast cancerspecimens fixed in ethanol at 4 (C overnight.

One of the tissue specimens that gave the fluorescent “positive” signalswas also analyzed by immunoperoxidase staining, as shown in FIG. 10D,where it was confirmed (by the dark stain) that the erbB2 gene productwas present. A DNA probe for the erbB2 gene was used to performfluorescent in situ hybridization (FISH). FIG. 10D shows one of thetumor array elements, which demonstrated high level erbB2 geneamplification. The insert in FIG. 10E shows three nuclei with numeroustightly clustered erbB2 hybridization signals and two copies of thecentromeric reference probe. Additional details about these assays aregiven in Examples 1-4 below.

The potential of the array technology of the present invention toperform rapid parallel molecular analysis of multiple tissue specimensis illustrated in FIGS. 11A-11D, where the y-axis of the graphs in FIGS.11A and 11C corresponds to percentages of tumors in specific groups thathave defined clinicopathological or molecular characteristics. Thisdiagram shows correlations between clinical and histopathologicalcharacteristics of the tissue specimens in the micro-array. Each smallbox in the aligned rows of FIG. 11B represents a coordinate location inthe array. Corresponding coordinates of consecutive thin sections of therecipient block are vertically aligned above one another in thehorizontally extending rows. These results show that the tissuespecimens could be classified into four classifications of tumors (FIG.11A) based on the presence or absence of cell membrane estrogen receptorexpression, and the presence or absence of the p53 mutation in thecellular DNA. In FIG. 11B, the presence of the p53 mutation is shown bya darkened box, while the presence of estrogen receptors is also shownby a darkened box. Categorization into each of four groups (ER−/p53+,ER−/p53−, ER+/p53+ and ER+/p53−) is shown by the dotted lines betweenFIGS. 11A and 11B, which divide the categories into Groups I, II, IIIand IV corresponding to the ER/p53 status.

FIG. 11B also shows clinical characteristics that were associated withthe tissue at each respective coordinate of the array. A darkened boxfor Age indicates that the patient is premenopausal, a darkened box Nindicates the presence of metastatic disease in the regional lymphnodes, a darkened box T indicates a stage 3 or 4 tumor which is moreclinically advanced, and a darkened box for grade indicates a high grade(at least grade III) tumor, which is associated with increasedmalignancy. The correlation of ER/p53 status can be performed bycomparing the top four lines of clinical indicator boxes (Age, N, T,Grade) with the middle two lines of boxes (ER/p53 status). The resultsof this cross correlation are shown in the bar graph of FIG. 11A, whereit can be seen that ER−/p53+ (Group I) tumors tend to be of higher gradethan the other tumors, and had a particularly high frequency of mycamplification, while ER+/p53+ (Group III) tumors were more likely tohave positive nodes at the time of surgical resection. The ER−/p53−(Group II) showed that the most common gene amplified in that group waserbB2. ER−/p53− (Group II) and ER+/p53− (Group IV) tumors, in contrast,were shown to have fewer indicators of severe disease, thus suggesting acorrelation between the absence of the p53 mutation and a betterprognosis.

This method was also used to analyze the copy numbers of several othermajor breast cancer oncogenes in the 372 arrayed primary breast cancerspecimens in consecutive FISH experiments, and those results were usedto ascertain correlations between the ER/p53 classifications and theexpression of these other oncogenes. These results were obtained byusing probes for each of the separate oncogenes, in successive sectionsof the recipient block, and comparing the results at correspondingcoordinates of the array. In FIG. 11B, a positive result for theamplification of the specific oncogene or marker (mybL2, 20q13, 17q23,myc, cnd1 and erbB2) is indicated by a darkened box. The erbB2 oncogenewas amplified in 18% of the 372 arrayed specimens, myc in 25% and cyclinD1 (cnd1) in 24% of the tumors.

The two recently discovered novel regions of frequent DNA amplificationin breast cancer, 17q23 and 20q13, were found to be amplified in 13% and6% of the tumors, respectively. The oncogene mybL2 (which was recentlylocalized to 20q13.1 and found to be overexpressed in breast cancer celllines) was found to be amplified in 7% of the same set of tumors. MybL2was amplified in tumors with normal copy number of the main 20q13 locus,indicating that it may define an independently selected region ofamplification at 20q. Dotted lines between FIGS. 11B and 11C againdivide the complex co-amplification patterns of these genes into GroupsI-IV which correspond to ER−/p53+, ER−/p53−, ER+/p53+ and ER+/p53−.

FIGS. 11C and 11D show that 70% of the ER−/p53+ specimens were positivefor one or more of these oncogenes, and that myc was the predominantoncogene amplified in this group. In contrast, only 43% of the specimensin the ER+/p53− group showed co-amplification of one of these oncogenes,and this information could in turn be correlated with the clinicalparameters shown in FIG. 11A. Hence the microarray technology of thepresent invention permits a large number of tumor specimens to beconveniently and rapidly screened for these many characteristics, andanalyzed for patterns of gene expression that may be related to theclinical presentation of the patient and the molecular evolution of thedisease. In the absence of the microarray technology of the presentinvention, these correlations are more difficult to obtain.

A specific method of obtaining these correlations is illustrated in FIG.12, which is an enlargement of the right hand portion of FIG. 11B. Themicroarray 76 (FIG. 10A) is arranged in sections that contain seventeenrows and nine columns of circular locations that correspond tocross-sections of cylindrical tissue specimens from different tumors,wherein each location in the microarray can be represented by thecoordinates (row, column). For example, the specimens in the first rowof the first section have coordinate positions (1,1), (1,2) . . . (1,9),and the specimens in the second row have coordinate positions (2,1),(2,2), . . . , (2,9). Each of these array coordinates can be used tolocate tissue specimens from corresponding positions on sequentialsections of the recipient block, to identify tissue specimens of thearray that were cut from the same tissue cylinder.

FIG. 12 illustrates one conceptual approach to organizing and analyzingthe array, in which the rectangular array may be converted into a linearrepresentation in which each box of the linear representationcorresponds to a coordinate position of the array. Each of the lines ofboxes may be aligned so that each box that corresponds to an identicalarray coordinate position is located above other boxes from the samecoordinate position. Hence the boxes connected by dotted line 1correspond to the results that can be obtained by looking at the resultsat a coordinate position [for example (1,1)] in successive thin sectionsof the donor block, or clinical data that may not have been obtainedfrom the microarray, but which can be entered into the system to furtheridentify tissue from a tumor that corresponds to that coordinateposition. Similarly, the boxes connected by dotted line 10 correspond tothe results that can be found at coordinate position (2,1) of the array,and the boxes connected by dotted line 15 correspond to the results atcoordinate position (2,6) of the array. The letters a, b, c, d, e, f, g,and h correspond to successive sections of the donor block that are cutto form the array.

By comparing the aligned boxes along line 1 in FIG. 12, it can be seenthat a tumor was obtained from a postmenopausal woman with no metastaticdisease in her lymph nodes at the time of surgical resection, in whichthe tumor was less than stage 3, but in which the histology of the tumorwas at least Grade III. A tissue block was taken from this tumor and isassociated with the recipient array at coordinate position (1,1). Thisarray position was sectioned into eight parallel sections (a, b, c, d,e, f, g, and h) each of which contained a representative section of thecylindrical array. Each of these sections was analyzed with a differentprobe specific for a particular molecular attribute. In section a, theresults indicated that this tissue specimen was p53+; in section b thatit was ER−; in section c that it did not show amplification of the mybL2oncogene; in separate sections d, e, f, g and h that it was positive forthe amplification of 20q13, 17q23, myc, cnd1 and erbB2.

Similar comparisons of molecular characteristics of the tumor specimencylinder that was placed at coordinate position (2,1) can be made byfollowing vertical line 10 in FIG. 12, which connects the tenth box ineach line, and corresponds to the second row, first column (2,1) of thearray 76 in FIG. 10(A). Similarly the characteristics of the sections ofthe tumor specimen cylinder at coordinate position (2,6) can be analyzedby following vertical line 15 down through the 15^(th) box of each row.In this manner, parallel information about the separate sections of thearray can be performed for all 372 positions of the array. Thisinformation can be presented visually for analysis as in FIG. 12, orentered into a database for analysis and correlation of differentmolecular characteristics (such as patterns of oncogene amplification,and the correspondence of those patterns of amplification to clinicalpresentation of the tumor).

Analysis of consecutive sections from the tumor arrays enablesco-localization of hundreds of different DNA, RNA, protein or othertargets in the same cell populations in morphologically defined regionsof every tumor, which facilitates construction of a database of a largenumber of correlated genotypic or phenotypic characteristics ofuncultured human tumors. Scoring of mRNA in situ hybridizations orprotein immunohistochemical staining is also facilitated with tumortissue microarrays, because hundreds of specimens can be analyzed in asingle experiment. The tumor arrays also substantially reduce tissueconsumption, reagent use, and workload when compared with processingindividual conventional specimens one at a time for sectioning, stainingand scoring. The combined analysis of several DNA, RNA and proteintargets provides a powerful means for stratification of tumor specimensby virtue of their molecular characteristics. Such patterns will behelpful to detect previously unappreciated but important molecularfeatures of the tumors that may turn out to have diagnostic orprognostic utility.

Analysis techniques for observing and scoring the experiments performedon tissue array sections include a bright-field microscope, fluorescentmicroscope, confocal microscope, a digital imaging system based on a CCDcamera, or a photomultiplier or a scanner, such as those used in the DNAchip based analyses.

These results show that the very small cylinders used to prepare tissuearrays can in most cases provide accurate information, especially whenthe site for tissue sampling from the donor block is selected to containhistological structures that are most representative of tumor regions.It is also possible to collect samples from multiple histologicallydefined regions in a single donor tissue block to obtain a morecomprehensive representation of the original tissue, and to directlyanalyze the correlation between phenotype (tissue morphology) andgenotype. For example, an array could be constructed to include hundredsof tissues representing different stages of breast cancer progression(e.g. normal tissue, hyperplasia, atypical hyperplasia, intraductalcancer, invasive and metastatic cancer). The tissue array technologywould then be used to analyze the molecular events that correspond totumor progression.

A tighter packing of cylinders, and a larger recipient block can alsoprovide an even higher number of specimens per array. Entire archivesfrom pathology laboratories can be placed in replicate 500-1000 specimentissue microarrays for molecular profiling. Using automation of theprocedure for sampling and arraying, it is possible to make dozens ofreplicate tumor arrays, each providing hundreds of sections formolecular analyses. The same strategy and instrumentation developed fortumor arrays also enables the use of tissue cylinders for isolation ofhigh-molecular weight RNA and DNA from optimally fixed, morphologicallydefined tumor tissue elements, thereby allowing correlated analysis ofthe same tumors by molecular biological techniques (such as PCR-basedtechniques) based on RNA and DNA. When nucleic acid analysis is planned,the tissue specimen is preferably fixed (before embedding in paraffin)in an alcohol based fixative, such as ethanol or Molecular BiologyFixative (Streck Laboratories, Inc., Omaha, Nebr.) instead of informalin, because formalin can cross-link and otherwise damage nucleicacid. The tissue cylinder of the present invention provides an ampleamount of DNA or RNA on which to perform a variety of molecularanalyses.

The potential of this array technology has peen illustrated in FISHanalysis of gene amplifications in breast cancer. FISH is an excellentmethod for visualization and accurate detection of geneticrearrangements (amplifications, deletions or translocations) inindividual, morphologically defined cells. The combined tumor arraytechnology allows FISH to become a powerful, high-throughput method thatpermits the analysis of hundreds of specimens per day.

Automated high speed devices can also be used that incorporate the basicprinciples of the device described herein. Such devices can processmultiple donor and recipient trays or containers, and are described inProvisional Application Ser. No. 60/106,038 and PCT ApplicationUS99/04000, which are incorporated herein by reference. The devices arecontrolled by standard operating environments including a computer thatcomprises at least one high speed processing unit (CPU), in conjunctionwith a memory system, an input device, and one or more output devices.These elements are interconnected by at least one bus structure. The CPUis of familiar design and includes an ALU for performing computations, acollection of registers for temporary storage of data and instructions,and a control unit for controlling operation of the system. The CPU maybe a processor having any of a variety of architectures including Alphafrom Digital; MIPS from MIPS Technology, NEC, IDT, Siemens and others;x86 from Intel and others, including Cyrix, AMD, and Nexgen; 680x0 fromMotorola; and PowerPC from IBM and Motorola. For example, the inventioncould be implemented with a Power Macintosh 8500 available from AppleComputer, or an IBM compatible Personal Computer (PC).

Examples and Applications of Array Technologies

The automated tumor array technology easily allows testing of dozens orhundreds of markers from the same set of tumors. These studies can becarried out in a multi-center setting by sending replicate tumor arrayblocks or sections to other laboratories. The same approach would beparticularly valuable for testing newly discovered molecular markers fortheir diagnostic, prognostic, or therapeutic utility. The tissue arraytechnology also facilitates basic cancer research by providing aplatform for rapid profiling of hundreds or thousands of tumors at theDNA, RNA, and protein levels, leading to a construction of a correlateddatabase of biomarkers from a large collection of tumors. For example,search for amplification target genes requires correlated analyses ofamplification and expression of dozens of candidate genes and loci inthe same cell populations. Such extensive molecular analyses of adefined large series of tumors would be difficult to carry out withconventional technologies.

Applications of the tissue array technology are not limited to studiesof cancer, although the following Examples 1-4 disclose embodiments ofits use in connection with analysis of neoplasms. Array analysis couldalso be instrumental in understanding expression and dosage of multiplegenes in other diseases, as well as in normal human or animal tissues,including tissues from different transgenic animals or cultured cells.

Tissue arrays can also be used to perform further analysis on genes andtargets discovered from, for example, high-throughput genomics, such asDNA sequencing, DNA microarrays, or SAGE (Serial Analysis of GeneExpression) (Velculescu et al., Science, 270:484-487, 1995). Tissuearrays can also be used to evaluate reagents for cancer diagnostics, forinstance specific antibodies or probes that react with certain tissuesat different stages of cancer development, and to follow progression ofgenetic changes both in the same and in different cancer types, or indiseases other than cancer. Tissue arrays can be used to identify andanalyze prognostic markers or markers that predict therapy outcome forcancers. Tissue arrays compiled from hundreds of cancers derived frompatients with known outcomes permit one or more of DNA, RNA, and proteinassays to be performed on those arrays, to determine importantprognostic markers, or markers predicting therapy outcome.

Tissue arrays can also be used to help assess optimal therapy forparticular patients showing particular tumor marker profiles. Forexample, an array of tumors can be analyzed to determine which onesamplify and/or overexpress HER-2, such that the tumor type (or morespecifically the subject from whom the tumor was taken) would be a goodcandidate for anti-HER-2 Herceptin immunotherapy. In anotherapplication, tissue arrays can be used to find novel targets for genetherapy. For example, cDNA hybridization patterns (such as on a DNAchip) may reveal differential gene regulation in a tumor of a particulartissue type (such as lung cancer), or a particular histological sub-typeof the particular tumor (such as adenocarcinoma of the lung). Analysisof each of such gene candidates on a large tissue array containinghundreds of tumors would help determine which is the most promisingtarget for developing diagnostic, prognostic, or therapeutic approachesfor cancer.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Tissue Specimens

A total of 645 breast cancer specimens were used for construction of abreast cancer tumor tissue microarray. The samples included 372fresh-frozen ethanol-fixed tumors, as well as 273 formalin-fixed breastcancers, normal tissues and fixation controls. The subset of frozenbreast cancer samples was selected at random from the tumor bank of theinstitute of Pathology, University of Basel, which includes more than1500 frozen breast cancers obtained by surgical resections during1986-1997. Only the tumors from this tumor bank were used for molecularanalyses. This subset was reviewed by a pathologist, who determined thatthe specimens included 259 ductal, 52 lobular, 9 medullary, 6 mucinous,3 cribriform, 3 tubular, 2 papillary, 1 histiocytic, 1 clear cell, and 1lipid rich carcinoma. There were also 15 ductal carcinomas in situ, 2carcinosarcomas, 4 primary carcinomas that had received chemotherapybefore surgery, 8 recurrent tumors and 6 metastases.

Histological grading was performed only in invasive primary tumors thathad not undergone previous chemotherapy. Of these tumors, 24% were grade1, 40% grade 2, and 36% grade 3. The pT stage was pT1 in 29%, pT2 in54%, pT3 in 9%, and pT4 in 8%. Axillary lymph nodes had been examined in282 patients (45% pN0, 46% pN1, 9% pN2). All previously unfixed tumorswere fixed in cold ethanol at +4° C. overnight and then embedded inparaffin.

Example 2 Immunohistochemistry

After formation of the tissue array and sectioning of the donor block,standard indirect immunoperoxidase procedures were used forimmunohistochemistry (ABC-Elite, Vector Laboratories). Monoclonalantibodies from DAKO (Glostrup, Denmark) were used for detection of p53(DO-7, mouse, 1:200), erbB-2 (c-erbB-2, rabbit, 1:4000), and estrogenreceptor (ER ID5, mouse, 1:400). A microwave pretreatment was performedfor p53 (30 minutes at 90° C.) and erbB-2 antigen (60 minutes at 90° C.)retrieval. Diaminobenzidine was used as a chromogen. Tumors with knownpositivity were used as, positive controls. The primary antibody wasomitted for negative controls. Tumors were considered positive for ER orp53 if an unequivocal nuclear positivity was seen in at least 10% oftumor cells. The erbB-2 staining was subjectively graded into 3 groups:negative (no staining), weakly positive (weak membranous positivity),strongly positive (strong membranous positivity).

Example 3 Fluorescent In Situ Hybridization (FISH)

Two-color FISH hybridizations were performed using Spectrum-Orangelabeled cyclin D1, myc, or erbB2 probes together with corresponding FITClabeled centromeric reference probes (Vysis). One-color FISHhybridizations were done with spectrum orange-labeled 20q13 minimalcommon region (Vysis, and see Tanner et al., Cancer Res. 54:4257-4260(1994)), mybL2 and 17q23 probes (Barlund et al., Genes Chrom. Cancer20:372-376 (1997)). Before hybridization, tumor array sections weredeparaffinized, air dried and dehydrated in 70, 85, and 100% ethanolfollowed by denaturation for 5 minutes at 74° C. in 70% formamide-2×SSCsolution. The hybridization mixture contained 30 ng of each of theprobes and 15 μg of human Cot1-DNA. After overnight hybridization at 37°C. in a humidified chamber, slides were washed and counterstained with0.2 μM DAPI in an antifade solution. FISH signals were scored with aZeiss fluorescence microscope equipped with double-band pass filters forsimultaneous visualization of FITC and Spectrum Orange signals. Over 10FISH signals per cell or tight clusters of signals were considered asindicative of gene amplification.

Example 4 mRNA In Situ Hybridization

For mRNA in situ hybridization, tumor array sections were deparaffinizedand air dried before hybridization. Synthetic oligonucleotide probesdirected against erbB2 mRNA (Genbank accession number X03363,nucleotides 350-396) was labeled at the 3′-end with ³³P-dATP usingterminal deoxynucleotidyl transferase. Sections were hybridized in ahumidified chamber at 42° C. for 18 hours with 1×10⁷ CPM/ml of the probein 100 μL of hybridization mixture (50% formamide, 10% dextran sulfate,it sarkosyl, 0.02 M sodium phosphate, pH 7.0, 4×SSC, 1×Denhardt'ssolution and 10 mg/ml ssDNA). After hybridization, sections were washedseveral times in 1×SSC at 55° C. to remove unbound probe, and brieflydehydrated. Sections were exposed for three days to phosphorimagerscreens to visualize ERBB2 mRNA expression. Negative control sectionswere treated with RNase prior to hybridization, which abolished allhybridization signals.

The present method enables high throughput analysis of hundreds ofspecimens per array. This technology therefore provides an order ofmagnitude increase in the number of specimens that can be analyzed, ascompared to prior blocks where a few dozen individual formalin-fixedspecimens are in a less defined or undefined configuration, and used forantibody testing. Further advantages of the present invention includenegligible destruction of the original tissue blocks, and an optimizedfixation protocol which expands the utility of this technique tovisualization of DNA and RNA targets. The present method also permitsimproved procurement and distribution of human tumor tissues forresearch purposes. Entire archives of tens of thousands of existingformalin-fixed tissues from pathology laboratories can be placed in afew dozen high-density tissue microarrays to survey many kinds of tumortypes, as well as different stages of tumor progression. The tumor arraystrategy also allows testing of dozens or even hundreds of potentialprognostic or diagnostic molecular markers from the same set of tumors.Alternatively, the cylindrical tissue samples provide specimens that canbe used to isolate DNA and RNA for molecular analysis.

Example 5 Tissue Microarrays for Gene Amplification Surveys in ManyDifferent Tumor Types

To facilitate rapid screening for molecular alterations in manydifferent malignancies, a tissue microarray consisting of samples from17 different tumor types, from 397 individual tumors, were arrayed in asingle paraffin-block. Amplification of three oncogenes (CCND1, MYC,ERBB2) was analyzed in three Fluorescence in situ Hybridization (FISH)experiments from consecutive sections cut from the tissue microarray.Amplification of CCND1 was found in breast, lung, head and neck, andbladder cancer as well as in melanoma. ERBB2 was amplified in bladder,breast, colon, stomach, testis, and lung cancers. MYC was amplified inbreast, colon, kidney, lung, ovary, bladder, head and neck, andendometrial cancer.

The microarray was constructed from a total of 417 tissue samplesconsisting of 397 primary tumors from 17 different tumor types and 20normal tissues which were snap-frozen and stored at −70° C. Specimenswere fixed in cold ethanol (+4° C.) for 16 hours and then embedded inparaffin. An H&E-stained section was made from each block to definerepresentative tumor regions. Tissue cylinders with a diameter of 0.6 mmwere then punched from tumor areas of each “donor” tissue block andbrought into a recipient paraffin block using a custom-made precisioninstrument as described. Then 5 μm sections of the resulting multi-tumortissue microarray block were transferred to glass slides using theparaffin sectioning aid system (adhesive coated slides, (PSA-CS4x),adhesive tape, UV-lamp; Instrumedics Inc., New Jersey) supporting thecohesion of 0.6 mm array elements.

The primary tumors consisted of 96 breast tumors (41 ductal, 28 lobular,6 medullar, 5 mucinous, and 4 tubular carcinomas, 7 ductal carcinomas insitu (DCIS) and 5 phylloides tumors), 80 carcinomas of the lung (31squamous, 11 large cell, 2 small cell, 31 adeno, and 5bronchioloalveolar carcinomas), 17 head and neck tumors (12 squamouscell carcinomas of the oral cavity and 5 of the larynx), 32adenocarcinomas of the colon, 4 carcinoids (3 from the lung and one fromthe small intestine), 12 adenocarcinomas from the stomach, 28 clear cellrenal cell carcinomas, 20 testicular tumors (10 seminomas and 10terato-carcinomas), 37 transitional cell carcinomas of the urinarybladder (33 invasive (pT1-4) and 4 non-invasive tumors), 22 prostatecancers, 26 carcinomas of the ovary (12 serous, 12 endometroid, and 2mucinous tumors), 13 carcinomas from the endometrium, 3 carcinomas ofthe thyroid gland, 3 pheochromocytomas, and 4 melanomas. Normal tissuefrom breast, prostate, pancreas, small bowel, stomach, salivary gland,colon, and kidney were used as controls.

The tissue microarray sections were treated according to the ParaffinPretreatment Reagent Kit protocol (Vysis, Ill.) before hybridization.FISH was performed with Spectrum Orange-labeled CCND1, ERBB2, and MYCprobes. Spectrum Green-labeled centromeric probes CEP11 and CEP17 wereused as a reference (Vysis, Ill.). Hybridization and post-hybridizationwashes were according to the “LSI procedure” (Vysis, Ill.). Slides werethen counterstained with 125 ng/ml 4′,6-diamino-2-phenylindole inantifade solution. FISH signals were scored with a Zeiss fluorescencemicroscope equipped with double-band pass filters for simultaneousvisualization of Spectrum Green and Spectrum Orange signals (Vysis,Ill.). Amplification was defined as presence (in at least 5% of tumorcells) of either (a) more than 10 gene signals or tight clusters of atleast 5 gene signals; or (b) more than 3 times as many gene signals thancentromere signals of the respective chromosome.

Seventy-two amplifications were found in 968 successfully hybridizedtumor samples, whereas none of the normal tissues showed amplification.Amplification usually involved almost all tumor cells within an arrayelement. CCND1 amplification was found in 6 of 16 head and neckcarcinomas (38%), 14 of 62 breast carcinomas (23%), 1 of 6 DCIS (17%), 3of 27 bladder cancers (11%), 7 of 76 carcinomas of the lung (9%), and 1of 4 melanomas.

MYC amplification was observed in 2 of 11 endometrial cancers (18%), 9of 74 breast carcinomas (12%), 1 of 5 DCIS (20%), 1 of 17 head and neckcancers (6%), 1 of 22 tumors of the kidney (5%), 2 of 24 ovariancarcinomas (8%), 1 of 17 tumors of the testis (6%), 1 of 30 coloncarcinomas (3%), 7 of 78 lung tumors (9%), and in 1 of 33 bladder tumors(3%).

ERBB2 was amplified in 4 of 71 breast-carcinomas (6%), 4 of 6 DCIS(67%), 2 of 11 stomach cancers (18%), 1 of 30 colon carcinomas (3%), 1of 17 tumors of the testis (6%), and in 1 of 75 carcinomas of the lung(1%). Co-amplifications of all three genes were seen in two breastcarcinomas. Co-amplifications of two genes were found in two breastcarcinomas (CCND1/MYC and CCND1/ERBB2) and in one terato-carcinoma ofthe testis (MYC and ERBB2).

Consecutive sections cut from the block provide starting material forthe in situ detection of multiple DNA, RNA or protein targets in manytissues at a time, in a massively parallel fashion. The tissue arraytechnology permits increased capacity, automation, negligible damage tothe original tissue blocks from which the specimens are taken, theprecise positioning of tissue specimens, and the use of these tissues indifferent kinds of molecular analyses, besides immunostaining. It ispossible to retrieve 10-20 punched samples (or more) from each donorblock without significantly damaging it. This enables generation ofmultiple replicate array blocks, each with the identical coordinates,and the same specimens. The application of a precision instrument todeposit the samples in a predefined format also facilitates thedevelopment of automated image analysis strategies for the arrayedtumors. Depending on the thickness of the original tissue blocks,between 150 and 300 sections can be cut from each array block. Thistechnology enables analyses of even small primary tumors, therebypreserving often unique and precious tumor specimens for a large numberof analyses that may be of interest in future investigations.

The array data reported in this example agreed with the previousliterature on the presence or absence of gene amplification in 73% ofevaluations, although the number of samples per tumor type was too smallfor a comprehensive analysis of some tumor types in this pilot study.Previously described amplifications were not detected on the array in 9of 25 tumor types from which less than 25 samples were examined. Incontrast, when at least 25 cases were analyzed per tumor type, 92% ofthe known amplifications (11/12) were detected.

In this study, frozen tumor tissues were fixed in cold ethanol becausethis procedure allows the retention of good quality nucleic acids fromfixed tissue samples. Even formalin-fixed tumor tissues, such as thoseobtained at autopsy, can be analyzed by FISH for DNA copy numberalterations. However, the cold ethanol fixation is advantageous forFISH, because the samples require fewer pretreatments than samples fixedin 4% buffered formalin. Cold ethanol fixation may cause RNAs to degradein paraffin blocks after only a few months of storage, hence it may notbe desired to fix a large series of precious tissues in cold ethanol,unless RNA inhibitors are added or blocks stored in a manner thatprohibits this degradation.

Example 6 PDGFB FISH Experiments Using a Multi-Tumor Tissue Array

The multi-tumor tissue array of Example 5 was used in this experiment. Aplatelet derived growth factor beta (PDGFB) probe was obtained fromVysis Inc. of Downers Grove, Ill. The probe was obtained by PCRscreening of a genomic large-insert library using two sequence taggedsites (STS) in the gene sequence as a target for developing PCR primersthat were used in the PCR-based library screening. The hits obtainedfrom genomic library screening were further verified by their content ofthe STSs, as well as by hybridizing the probe to metaphase chromosomesusing FISH. This resulted in a signal at the expected chromosomallocation of PDGFB.

PCR/STS screening can be performed using a PCR primer set specific tothe gene of interest, as described by Green & Olson, PNAS USA,87:1213-1217, 1990. Probes for FISH may be generated from large-insertlibraries (e.g., cosmids, P1 clones, BACs, and PACs) using a PCR-basedscreening of arrayed and pooled large-insert libraries. Both ResearchGenetics (Huntsville, Ala.) and Genome Systems (St. Louis) perform suchfilter screening, and sell pools of DNA for performing libraryscreening.

One method of isolating the P1 clone for PDGFB (pVYS309A) would be toscreen DNA pools of a human P1 library obtained from Genome Systems,Inc. Individual clones are identified by producing the expected DNAfragment size on gels after PCR. Bacterial cultures containing candidatePDGFB clones are purified by streaking on nutrient agar media for singlecolonies. Cultures from individual colonies are then grown and DNAisolated by standard techniques. The DNA is confirmed to contain thedesired DNA sequence by PCR and gel electrophoresis (STS confirmation).A sample of the DNA is labeled by nick-translation or random primingwith SpectrumOrange dUTP (Vysis) and shown to hybridize to the expectedregion of chromosome 22q normal metaphase chromosomes by FISH.

PCR primers for PDGFB can be derived from the published sequence of thecDNA of this gene (GenBank Accession X02811). The preferred region ofSTS design is the 3′ untranslated region of the cDNA. Several PCR primersets for PDGFB are in public databases, e.g., amplimers (PCR primersets) PDGFB PCR1, PDGFB PCR2, PDGFB PCR3, stPDGFB.b, WI-8985, and can befound in the Genome Database (http://gdbwww.gdb.org/gdb/gdbtop.html).WI-8985 primer sets can also be found at the Whitehead Institutedatabase (http://www-genome.wi.mit.edu/), and at the NIH Gene Map 98database (http://www.ncbi.nlm.nih.gov/genemap98/).

FISH was done using standard protocols, as in Example 5, andhybridization of the probe to specimens of the tissue array was detectedas in Example 5. Hybridization was detected in the following types oftumors:

TUMOR Ratio Positive Percent Positive breast CA  2/70 2.9% phylloides0/4 DCIS 0/7 lung 15/77  19% colon  1/30 3.3% carcinoid 0/3 stomach 0/9renal cell  0/11 testis  1/16   6% TCC 10/32  31% (bladder transitionalcell carcinoma) head/neck  0/17 PCA  0/18 ovary  0/22 endometrium 2/8 25% Total  22/324

This Example provides the first evidence of previously unsuspected,high-level amplifications of PDGFB in specific types of malignancies,such as breast, lung, colon, testicular, endometrial, and bladdercancer.

Example 7 Gene Amplifications During Prostate Cancer Progression

In this study, five different gene amplifications (AR, CMYC, ERBB2,Cyclin D1, and NMYC) were assayed by FISH from consecutive formalinfixed tissue microarray sections containing samples from more than 300different prostate tumors. The objective was to obtain a comprehensivesurvey of gene amplifications in different stages of prostate cancerprogression, including specimens from distant metastases. The tissuemicroarray contained minute samples from 371 specimens.

Formalin-fixed and paraffin-embedded tumor and control specimens wereobtained from the archives of the Institutes for Pathology, Universityof Basel (Switzerland) and the Tampere University Hospital (Finland).The least differentiated tumor area was selected to be sampled for thetissue microarray. The minute specimens that were interpretable for atleast one FISH probe included: I) transurethral resections from 32patients with benign prostatic hyperplasia (BPH) which were used ascontrols; II) 223 primary tumors, including 64 cancers incidentallydetected in transurethral resections for BPH; stage T1a/b, 145clinically localized cancers from radical prostatectomies, and 14transurethral resections from patients with primary; locally extensivedisease; III) 54 local recurrences after hormonal therapy failureincluding 31 transurethral resections from living patients and 23specimens obtained from autopsies; IV) Sixty-two metastases collected atthe autopsies from 47 patients who had undergone androgen deprivation byorchiectomy, and had subsequently died of end-stage metastatic prostatecancer. Metastatic tissue was sampled from pelvic lymph nodes (8), lung(21), liver (16), pleura (5), adrenal gland (5), kidney (2), mediastinallymph nodes (1), peritoneum (1), stomach (1), and ureter (1). In 23autopsies material was available from both the primary and from themetastatic site. More than one sample per tumor specimen was arrayed in44 of the 339 cases. A tumor was considered amplified if at least onesample from the tumor exhibited gene amplification.

The array also included 48 pathologically representative samples whichconsistently failed in the analysis of sections with all FISH probes,and were therefore excluded from the analyses. Most of these wereautopsy samples. The number of samples evaluated with the differentprobes was variable, because the hybridization efficiency of the probeswas slightly different, some samples on the array were occasionally lostduring the sectioning or FISH-procedure, and some tumors were onlyrepresentative on the surface of the block, and the morphology changedas more sections were cut.

The prostate tissue microarray was constructed as previously describedin Example 1, except with prostate instead of breast cancer specimens.

Two-color FISH to sections of the arrayed formalin-fixed samples wasperformed using Spectrum orange-labeled AR, CMYC, ERBB2, and CyclinD1(CCND1) probes with corresponding FITC-labeled centromeric probes(Vysis, Downer's Grove, Ill.). In addition, one-color FISH was done witha Spectrum Orange-labeled NMYC probe (Vysis). The hybridization wasperformed according to the manufacturer's instructions. To allowformalin-fixed tumors on the array to be reliably analyzed by FISH, theslides of the prostate microarray were first deparaffinized, acetylatedin 0.2 N HCl, incubated in 1 M sodium thiocyanate solution at 80° C. for30 minutes and immersed in a protease solution (0.5 mg/ml in 0.9% NaCl)(Vysis) for 10 minutes at 37° C. The slides were then post-fixed in 10%buffered formalin for 10 minutes, air dried, denatured for 5 minutes at73° C. in 70% formamide/2×SSC (SSC is 0.3 M sodium chloride and 0.03 Msodium citrate) solution and dehydrated in 70, 80, and 100% ethanol,followed by proteinase K (4 μg/ml phosphate buffered saline) (GIBCOBRL,Life Technologies Inc., Rockville, Md.) treatment for 7 minutes at 37°C. The slides were then dehydrated and hybridized.

The hybridization mixture contained 3 μl of each of the probes andCot1-DNA (1 mg/ml; GIBCOBRL, LifeTechnologies Inc., Rockville, Md.) in ahybridization mixture. After overnight hybridization at 37° C. in ahumid chamber, slides were washed, and counterstained with 0.2 μM DAPI.FISH signals were scored with a Zeiss fluorescence microscope equippedwith a double-band pass filter using x40-x100 objectives. The relativenumber of gene signals in relation to the centromeric signals wasevaluated. Criteria for gene amplification were: at least 3 times moretest probe signals than centromeric signals per cell in at least 10% ofthe tumor cells. Test/control signal ratios in the range between 1 and 3were regarded as low level gains, and were not scored as evidence ofspecific gene amplification. Amplification of NMYC without a referenceprobe was defined as at least 5 gene signals in at least 10% of thetumor cells.

High-quality hybridization signals with both centromeric and genespecific probes were obtained in 96% of the BPH samples for chromosomeX/AR gene, 84% for chromosome 8/CMYC, 81% for chromosome 17/ERBB2, and83% for chromosome 11/Cyclin D1. In the BPH samples that could beevaluated, the average percentage of epithelial cells with two signalsfor autosomal probes was ˜75%, with ˜20% showing one signal, and ˜5% nosignals. The percentage of cells with one or zero signals is believed tobe attributable to the truncation of nuclei with sectioning. In thepunched (single array element) samples of biopsy cancer specimens, AR,CMYC, ERBB2, and CCND1 FISH data could be obtained from 92%, 78%, 82%,and 86% of the cases, respectively. The success rate of FISH was lowerin punches from autopsy tumors (44-58%). Amplifications were only scoredwhen the copy number of the test probe exceeded that of thechromosome-specific centromere reference probe by ≧3-fold in 10% or moreof the tumor cells. This criterion was chosen, as low-levelamplification is likely to be less relevant, and since locus-specificprobes often display slightly higher copy numbers than centromericprobes, due to signal splitting or the presence of G2/M-phase cells.

FISH with the AR probe revealed amplification in 23.4% of the 47evaluable hormone-refractory local recurrences. Amplification was seenequally often (22.0%) in 59 metastases of hormone-refractory tumors. Thestrong association between AR amplification and hormone-refractoryprostate cancer is evident from the fact that only two of the 205evaluable primary tumors (1%) and none of the 32 BPH controls showed anyAR amplification. The two exceptions included a patient with locallyadvanced and metastatic prostate cancer, and another patient withclinically localized disease. Paired tumors from the primary site of thecancer and from a distant metastasis of 17 patients were successfullyanalyzed for AR amplification. In 11 of these patients, no ARamplification could be seen at either site. Of the six remainingpatients, three patients showed amplification both in the local tumormass, as well as in the distant metastases. In two cases amplificationwas only found in the sample from the primary site, whereas in anothercase only the distant metastasis showed amplification.

High-level CMYC amplifications were found in 5 of 47 evaluablemetastatic deposits (10.6%), in 2 of the 47 local recurrences (4.3%,both metastatic cancers), but in none of the 168 evaluable primarycancers or 31 BPH controls. The comparison between different geneamplifications within the tumor cells defined by single punch-samples(array elements) showed that there was a significant association betweenAR and CMYC amplifications. CMYC was amplified in 11.1% of 27 evaluablepunch-samples with AR amplifications but only in 1.7% of 235 sampleswithout AR amplifications (p=0.0041, contingency table analysis). AR wasindependently amplified in 24 samples, whereas only four samples hadCMYC amplification, but no AR amplification.

On a tumor by tumor basis, there was a significant association betweenAR and CMYC amplifications. CMYC was amplified in 12.5% of 24 evaluabletumors with AR amplifications, but only in 1.8% of 219 tumors without ARamplifications (p=0.003, contingency table analysis). AR wasindependently amplified in 21 tumors, whereas only 4 tumors had CMYCamplification, but no AR amplification.

CCND1 amplifications were found in 2 (1.2%) of the 172 evaluable primarytumors, in 3 (7.9%) of 38 local recurrences, and in 2 (4.7%) of the 43metastases. CCND1 amplification appeared independent from AR or CMYCamplification with 4/7 CCND1 amplified punched tumor samples hot showingamplifications for any other genes tested. There were no ERBB2amplifications among any of the 262 evaluable tumors or 31 BPH controls.Finally, a subset of the tumors was analyzed with the NMYC probe in asingle color FISH analysis. Out of the 164 tumors available, none showedamplification, as defined by the lack of 5 or more signals per cellin >10% of the tumor cells.

For this study a tumor array was constructed that allowed investigationof the pattern of amplifications of multiple genes in samplesrepresenting the entire spectrum of prostate cancer progression,including distant metastases. The tumor array strategy facilitatesstandardized analysis of multiple genes in the same tumors, even in thesame specific tumor sites using the same technology, with the same kindof probes, and similar interpretation criteria. In just five FISHexperiments, 371 specimens were screened for five genes resulting in atotal of over 1400 evaluable FISH results. The ability to achievereliable detection of gene amplifications from formalin-fixed tissuessubstantially extends the range of possible applications for the tumorarray technology.

Many symptomatic prostate cancers become both hormone-refractory andmetastatic, and it is difficult to distinguish between these twoclinical features, or the molecular mechanisms that contribute to eitherof these processes. The results of the present example indicate that ARamplification is more closely associated with the development ofhormone-refractory cell growth, whereas CMYC amplification is associatedwith metastatic progression. The most common gene amplification inprostate cancers is that of the AR gene, which is usually amplifiedindependently of both CMYC and Cyclin D1. In this study, CMYCamplifications were more common in the distant metastases (11%) than inthe locally recurrent tissues (4%; both from patients with end-stagemetastatic cancers), whereas AR amplifications were equally common atboth anatomical sites (22% and 23%, respectively). This suggests that ARis conferring an advantage for hormone-refractory growth, and notmetastatic dissemination, whereas the reverse may be true for CMYC.

This Example indicates that the AR gene is the most frequent target, andoften the first target, selected for amplification during prostatecancer progression. Second, in contrast to AR, amplifications of theCMYC oncogene appear to be primarily associated with metastaticdissemination. Finally, prostate cancers occasionally also amplify theCyclin D1 gene, whereas ERBB2 and NMYC amplifications are unlikely toplay a significant role at any stage of the progression of prostatecancer.

Example 8 Rapid Screening for Prognostic Markers in Renal CellCarcinomas (RCC) by Combining cDNA-Array and Tumor-Array Technologies

This example first uses cDNA arrays to identify genes that play a rolein renal cell carcinoma (RCC), and subsequently analyzes emergingcandidate genes on a tumor array for their potential clinicalsignificance. The results show that the combination of nucleic acidarrays and tumor arrays is a powerful approach to rapidly identify andfurther evaluate genes that play a role in RCC biology.

cDNA was synthesized and radioactively labeled using 50 μg of total RNAfrom normal kidney (Invitrogen) and a renal cancer cell line (CRL-1933)(ATCC, VA, USA) according to standardized protocols (Research Genetics;Huntsville, Ala.). Release I of the human GeneFilters from ResearchGenetics was used for differential expression screening. A singlemembrane contained 5184 spots each representing 5 ng of cDNA of knowngenes or expressed sequence tags (EST's). After separate hybridizationthe two cDNA array filters (Research Genetics) were exposed to a highresolution screen (Packard) for three days. The gene expression patternof 5184 genes in normal tissue and the tumor cell line was analyzed andcompared on a phosphor imager (Cyclone, Packard). To define genes/EST'sas under- or overexpressed, both an at least tenfold expressiondifference between normal tissue and the cell line using the Pathfindersoftware (Research Genetics; Huntsville, Ala.) and visual confirmationof an unequivocal difference in the staining intensity on filters wasrequested.

For the construction of the renal tumor microarray block, a collectionof 615 renal tumors after nephrectomy was screened for availability ofrepresentative paraffin-embedded tissue specimens. Tumor specimens from532 renal tumors and tissue from 6 normal kidneys were selected for thetumor array. The tumors were staged according to TNM classification,graded according to Thoenes (Pathol. Res. Pract., 181:125-143, 1986) andhistologically subtyped according to the recommendations of the UICC(Bostwick et al., Cancer, 80:973-1001, 1997) by one pathologist.Core-tissue-biopsies (diameter 0.6 mm) were taken from selectedmorphologically representative regions of individual paraffin-embeddedrenal tumors (donor blocks) and precisely arrayed into a new recipientparaffin block (45 mm×20 mm) using a custom-built instrument. Then 5 μmsections of the resulting tumor tissue micro array block weretransferred to glass slides using the paraffin sectioning aid system(Adhesive coated slides, (PSA-CS4x), adhesive tape, UV-lamp;Instrumedics Inc., New Jersey) supporting the cohesion of 0.6 mm arrayelements.

Standard indirect immunoperoxidase procedures were used forimmunohistochemistry (ABC-Elite, Vectra Laboratories) as described, forexample in Moch et al., Hum. Pathol., 28:1255-1259, 1997. A monoclonalantibody was employed for vimentin detection (anti-vimentin; BoehringerMannheim, Germany, 1:160). Tumors were considered positive for vimentin,if an unequivocal cytoplasmic positivity was seen in tumor cells.Vimentin positivity in endothelial cells served as an internal control.The vimentin positivity in epithelial cells was defined as negative (nostaining) or positive (any cytoplasmic staining).

Contingency table analysis was used to analyze the relationship betweenvimentin expression, grade, stage, and tumor type. Overall survival wasdefined as the time between nephrectomy and patient death. Survivalrates were plotted using the Kaplan-Meier method. Survival differencesbetween the groups were determined with the log-rank test. A Coxproportional hazard analysis was used to test for independent prognosticinformation.

Two cDNA array membranes were hybridized with radioactive-labeled cDNAfrom normal kidney and tumor cell line CRL-1933. The experiment resultedin 89 differentially expressed genes/EST's. An overexpression inCRL-1933 was found for 38 sequences, including 26 named genes and 12EST's while 51 sequences (25 named genes, 26 EST's) were underexpressedin the cell line. The sequence of one of the upregulated genes in thecell line was identical to vimentin.

The presence of epithelial tumor cells was tested for every tissuecylinder using an H&E-stained slide. Vimentin expression could beevaluated on the tissue cylinders in 483 tumors and all 6 normal kidneytissues. Vimentin expression was frequent in clear-cell (51%) andpapillary RCC (61%), but rare in 23 chromophobe RCC (4%). Only 2 of 17oncocytomas showed a weak vimentin expression (12%). Normal renaltubules did not express vimentin. The association between vimentinexpression and histological grade and tumor stage was only evaluated forclear cell RCC. Vimentin expression was more frequent in grade II (44%)and grade III (42%) than in grade I (13%) RCC (p<0.0001). Vimentinexpression was more common in higher tumor stages (60% in stage pT1/2versus 40% in stage pT3/4), but this difference was not significant(p=0.09).

There was a mean follow-up of 52.9±51.4 months (median, 37, minimum 0.1,maximum 241 months). Poor overall survival was strongly related to highhistologic grade (p<0.0001) and high tumor stage (p<0.0001). Theassociation between patient prognosis and vimentin expression wasevaluated for patients with clear cell RCC. Vimentin expression wasstrongly associated with short overall survival (p=0.007). ProportionalHazards analysis with the variables tumor stage, histological grade, andvimentin expression indicates that vimentin expression was anindependent predictor of prognosis, the relative risk being 1.6 (p=0.01)in clear cell RCC.

The results of this example show that the combination of cDNA and tumorarrays is a powerful approach for identification and further evaluationof genes playing a role in human malignancies. This example illustratesthat cDNA arrays may be used to search for genes that are differentiallyexpressed in tumor cells (such as kidney cancer) as compared to normaltissue (kidney tissue in this example). Evaluation of all candidategenes emerging from a cDNA experiment on a representative set ofuncultured primary tumors would take years if traditional methods ofmolecular pathology were used. However the tumor microarray technologymarkedly facilitates such studies. Tissue arrays allow the simultaneousin situ analysis of hundreds of tumors on the DNA, RNA and proteinlevel, and even permits correlation with clinical follow up data.

This high throughput analysis allowed marked differences in the vimentinexpression between renal tumor subtypes to be illustrated. Vimentin wasfrequently detected in papillary and clear cell RCC, but rarely inoncocytoma and chromophobe RCC. Given the high rate of vimentinpositivity in clear cell RCC detected in this example, the presence ofvimentin expression may be used as a diagnostic feature to distinguish adiagnosis of clear cell RCC from chromophobe RCC.

This example further illustrates that tumor tissue arrays can facilitatethe translation of findings from basic research into clinicalapplications. The speed of analysis permits a multi-step strategy.First, molecular markers or genes of interest are assessed on a mastermulti-tumor-array containing samples of many (or all) possible humantumor type. In a second step, all tumor types that have shownalterations in the initial experiment are then further examined on tumortype-specific arrays (for example bladder cancer) containing much highernumbers of tumors of the same tissue type, with clinical follow upinformation on survival or response to specific therapies. In a thirdstep the analysis of conventional (large) diagnostic histologic andcytologic specimens is then restricted to those markers for whichpromising data emerged during the initial array based analyses. Forexample, vimentin expression can now be studied on larger tissuespecimens to confirm its prognostic significance in clear cell RCC. Ifthe array data are confirmed, vimentin immunohistochemistry may then beincluded in prospective studies investigating prognostic markers in RCC.

Example 9 DNA Array Technology

Instead of using a single probe to test for a specific sequence on thesample DNA, a gene or DNA chip incorporates many different “probes.”Although a “probe” usually refers to what is being labeled andhybridized to a target, in this situation the probes are attached to asubstrate. Many copies of a single type of probe are bound to the chipsurface in a small spot which may be, for example, approximately 0.1 mmor less in diameter. The probe may be of many types including DNA, RNA,cDNA, or oligonucleotide. In variations of the technology, specificproteins, polypeptides or immunoglobulins or other natural or syntheticmolecules may be used as a target for analyzing DNA, RNA, protein orother constituents of cells, tissues, or other biological specimens.Many spots, each containing a different molecular target, are thenarrayed in the shape of a grid. The surface for arraying may be a glass,or other solid material, or a filter paper or other related substanceuseful for attaching biomolecules. When interrogated with labeledsample, the chip indicates the presence or absence of many differentsequences or molecules in that specimen. For example, a labeled cDNAisolated from a tissue can be applied on a DNA chip to assay forexpression of many different genes at a time.

The power of these chips resides not only in the number of differentsequences or other biomolecules that can be probed simultaneously, asexplained below for nucleic acid chips. In the analysis of nucleicacids, a relatively small amount of sample nucleic acid is required forsuch an analysis (typically less than a millionth of a gram of nucleicacid). The binding of nucleic acid to the chip can be visualized byfirst labeling the sample nucleic acid with fluorescent molecules or aradioactive label. The emitted fluorescent light or radioactivity can bedetected by very sensitive cameras, confocal scanners, image analysisdevices, radioactive film or a PHOSPHOIMAGER™, which capture the signals(such as the color image) from the chip. A computer with image analysissoftware detects this image, and analyzes the intensity of the signalfor each probe location in the array. Detection of differential geneexpression with a radioactive cDNA array was already described inExample 8. Usually, signals from a test array are compared with areference (such as a normal sample).

DNA chips may vary significantly in their structure, composition, andintended functionality, but a common feature is usually the small sizeof the probe array, typically on the order of a square centimeter orless. Such an area is large enough to contain over 2,500 individualprobe spots, if each spot has a diameter of 0.1 mm and spots areseparated by 0.1 mm from each other. A two-fold reduction in spotdiameter and separation can allow for 10,000 such spots in the samearray, and an additional halving of these dimensions would allow for40,000 spots. Using microfabrication technologies, such asphotolithography, pioneered by the computer industry, spot sizes of lessthan 0.01 mm are feasible, potentially, providing for over a quarter ofa million different probe sites.

Targets on the array may be made of oligomers or longer fragments ofDNA. Oligomers, containing between 8 and 20 nucleotides, can besynthesized readily by chemical methods. Photolithographic techniquesallow the synthesis of hundreds of thousands of different types ofoligomers to be separated into individual spots on a single chip, in aprocess referred to as in situ synthesis. Long pieces of DNA, on theother hand, contain up to several thousand nucleotides, and can not besynthesized through chemical methods. Instead, they are excised from thehuman genome and inserted into bacterial cells through geneticengineering techniques. These cells, or clones, serve as a convenientsource for these DNAs, which can be produced in large quantities byfermentation. After extraction and appropriate chemical preparation theDNA from each clone is deposited onto the chip by a robot, which isequipped either with very fine syringes or with an ink-jet system.

The targets on the DNA chip interact with the DNA that is being analyzed(the target DNA) by hybridizing. The specificity of this process (theaccuracy with which the sample nucleic acid sequences will bind to theircomplementary arrayed target sequences) is mainly a function of thelength of the probe. For short oligonucleotide probes, the conditionscan be chosen such that a single point mutation (the change of a singlenucleotide in a gene) can be detected. That may require as many as65,536 or even more different oligonucleotide probes on a single chip tounambiguously deduce the sequence of even a relatively small DNAsequence. This process, called sequencing by hybridization (SbH),generates very complex hybridization patterns that are interpreted byimage analysis computer software. In addition, the sequence to beanalyzed is preferably short, and it must be isolated and amplified fromthe rest of the genome through a technique called Polymerase ChainReaction (PCR), before it is applied to the chip for sequence analysis

In Comparative Genomic Hybridization (CGH), DNA from a sample tissue,such as a tumor, is compared to normal human DNA. In a particularexample of CGH performed by Vysis, Inc., this is accomplished bylabeling the sample DNA with a fluorescent dye, and the reference(“normal”) DNA with a fluorescent dye of a different color. Both DNAsare then mixed in equal amounts and hybridized to a DNA chip. The Vysischip or genosensor, contains an array of large insert DNA clones, eachcomprising approximately 100,000 nucleotides of human DNA sequence.After hybridization, a multi-color imaging system determines the ratioof colors (for example green to red fluorescence) for each of the probespots in the array. If there is no difference between the sample DNA andthe normal DNA, then all spots should have an equal mixture of red andgreen fluorescence, resulting in a yellow color. A shift toward green orred for a given spot would indicate that either more green or more redlabeled DNA was bound to the chip by that probe sequence. This colorshift indicates a difference between the sample and the reference DNAfor that particular region on the human genome, pointing either towardamplification or deletion of a specific sequence or gene contained inthe clones positioned in the array. Examples of genetic changes that canbe detected include amplifications of genes in cancer, or characteristicdeletions in genetic syndromes, such as Cri du chat.

Since each genetic region to be analyzed need to be represented on thechip in only 1 or few replicate spots, the genosensor can be designed toscan the total human genome for large deletions or duplications in asingle assay. For example, an array of just 3000 different clones evenlyspaced along the human genome would provide a level of resolution thatis at least 10 times better than what can be achieved with metaphasehybridization, at a much lower cost and in much less time. Specialtychips can be tailored to the analysis of certain cancers or diseasesyndromes, and can also provide physicians with much more information onroutine clinical analysis than currently can be obtained even by themost sophisticated research laboratories.

The color ratio analysis of the genosensor CGH (gCGH) assay has theadvantage that absolute quantitation of the amount of a specificsequence in the sample DNA is not required. Instead, only the relativeamount compared to the reference (normal) DNA is measured withrelatively high accuracy. This approach is equally useful for a thirdkind of chip technology, referred to as “Expression Chips.” These chipscontain arrays of probe spots which are specific for different genes inthe human genome. They do not measure the presence or absence of amutation in the DNA directly, but rather determine the amount of messagethat is produced from a given gene. The message, or mRNA, is anintermediary molecule in the process by which the genetic informationencoded in the DNA is translated into protein. The process by which mRNAamounts are measured involves an enzymatic step which converts theunstable mRNA into cDNA, and simultaneously incorporates a fluorescentlabel. cDNA from a sample tissue is labeled in one color and cDNA from anormal tissue is labeled with a different color. After comparativehybridization to the chip, a color ratio analysis of each probe spotreveals the relative amounts of that specific mRNA in the sample tissuecompared to normal tissue. Expression chips measure the relativeexpression of each gene for which there is a probe spot on the chip.

There are approximately 100,000 different genes in the human genome, andit is expected that all of them will be known within a few years. Sincechips with thousands of different probe spots can be made, the relativeexpression of each gene can be determined in a single assay. This hassignificant implications for disease diagnosis and therapy. Expressionchips may be used to test the effect of drugs on the expression of alimited number of genes in tissue culture cells, by comparing mRNA fromdrug treated cells to that of untreated cells. The ability to measurethe effect on the regulation of all genes will allow a much more rapidand precise drug design, since the potency and potential side effects ofdrugs can be tested early in development. Moreover, the rapid increasein understanding of the regulatory switches that determine tissuedifferentiation will allow for the design of drugs that can initiate ormodify these processes. Findings about differential expression in CGHcan be further analyzed in tissue arrays, in which expression of mRNAcan also be determined.

In one particular embodiment of CGH, a DNA chip or genosensor (hence,genosensor CGH or gCGH), such as an AmpliOnc™ chip from Vysis, containsan array of P1, BAC, or PAC clones, each with an insert of human genomicDNA. The size of these inserts ranges from 80 to 150 kilobases, and theyare spaced along the human genome to improve the resolution of thistechnique. Since the hybridization probe mixture contains only on theorder of 200 ng of total human DNA from each of the test and referencetissue, the total number of available probes for each arrayed targetclone is relatively low, placing higher demands on the sensitivity ofthis system than what is needed for regular fluorescent in situhybridization techniques. These demands have been met with thedevelopment of improved chip surfaces, attachment chemistry, and imagingsystems. The combination of such features can provide a sensitivity of<10⁸ fluorophors/cm², which is achieved through highly efficientbackground reduction.

Autofluorescence emanating from the chip surface may be reduced bycoating the glass chip with chromium, as disclosed in U.S. patentapplication Ser. No. 09/085,625. This highly reflective surface providesenhanced signal collection efficiency, and its hydrophobic naturereduces non-specific binding of probes. Efficient reading of CGH chipsis achieved with a sensitive, high speed, compact, and easy to usemulticolor fluorescence imaging system, such as that described in U.S.patent application Ser. No. 09/049,748. The non-epifluorescentexcitation geometry eliminates autofluorescence from the collectionoptics, and collects only fluorescent light from the chip surface. Axenon arc lamp serves as a safe and long-lasting light source, providingeven illumination over a wide range of wavelengths. This allows for theuse of many different fluorophores, limited only by the choice ofexcitation and emission filters. Fluorescent images are acquired from a14 mm×9 mm sample area by a cooled CCD camera without scanning ormagnification, and even the need for routine focusing has beeneliminated. The images are analyzed by software, which interrogates eachindividual pixel to calculate the ratio of sample to reference probethat are hybridized to each target spot. An appropriate statisticalanalysis reveals the relative concentration of each target specificsequence in the probe mixture.

This system may be used for expression analysis or genomic applications,such as an analysis of genetic changes in cancer. For this purpose amicroarray was developed for the specific analysis of all geneticregions that have been reported so far to be associated with tumorformation through amplification at the genome level. The AmpliOnc™ chipcontains 33 targets (mostly known oncogenes), each replicated 5 times. Aschematic representation of such a chip (and 31 of the targets) is shownin FIG. 13. New chips containing 50 targets or more can also be used.

Example 10 Combination of Microarrays to Detect Amplification of FGFR2Gene in Sum-52 Breast Cancer Cell Line

This Example demonstrates how target genes for chromosomal gains seen bycomparative genomic hybridization (CGH) can be rapidly identified andstudied for their clinical relevance using a combination of novel,high-throughput microarray strategies. CGH to metaphase spreads (FIG.14, chromosomal CGH) showed high-level DNA amplifications at chromosomalregions 7q31, 8p11-p12 and 10q25 in the Sum-52 breast cancer cell line.Genomic DNA from the Sum-52 cell line was then hybridized to a novel CGHmicroarray (FIG. 14, genosensor CGH, Vysis, Downers Grove, Ill.), whichenabled simultaneous screening of copy number ac 31 loci containingknown or suspected oncogenes (the loci are shown in FIG. 13). This gCGHanalysis implicated specific, high-level amplifications of the MET (at7q31) and FGFR2 (at 10q25) genes, as well as low level amplification ofthe FGFR1 gene (at 8p11-p12), indicating the involvement of these threegenes in the amplicons seen by conventional CGH analysis.

A large-scale expression survey of the same cell line using a cDNAmicroarray (Clonetech Inc.) provided additional information. The FGFR2gene was the most abundantly overexpressed transcript in the SUM-52cells implicating this gene as the likely amplification target gene at10q25. Overexpression of FGFR2 was confirmed by Northern analysis, andamplification by fluorescence in situ hybridization (FISH). Finally,FISH to a tissue microarray consisting of 145 primary breast cancers(FIG. 15) showed the in vivo amplification of the FGFR2 gene in 4.5% ofthe cases.

These three microarray experiments can be accomplished in a few days,and illustrate how the combination of microarray-based screeningtechniques is very powerful for the rapid identification of target genesfor chromosomal rearrangements, as well as for the evaluation of theprevalence of such alterations in large numbers of primary tumors. Thispower is conferred by the ability to screen many genes against onetumor, using DNA array technologies (such as cDNA chips or CGH), to finda gene of interest, in combination with the ability to screen manytumors against the gene of interest using the tissue microarraytechnology. FIG. 16 illustrates that the DNA chip can use multipleclones (for example more than 100 clones) to screen a single tumor orother cell, while the complementary tissue microarray technology can usea single probe to screen multiple (for example more than 100) tumor orother tissue specimens (of either the same or different tissue types).

Example 11 Tissue Arrays to Determine Frequency and Distribution of GeneExpression and Copy Number Chances During Cancer Progression

Tissue arrays may be used to follow up genes and targets discoveredfrom, for example, high-throughput genomics, such as DNA sequencing, DNAmicroarrays, or SAGE (Serial Analysis of Gene Expression) (Velculescu etal., Science, 270:484-487, 1995). Comparative analysis of geneexpression patterns with cDNA array technology (Schena 1995 and 1996)provides a high-throughput tool for screening expressional changes forbetter understanding molecular mechanisms responsible for tumorprogression as well as aiming for discovery of new prognostic markersand potential therapeutic targets. Tissue arrays provide accuratefrequency and distribution information concerning such genes in bothpathological and normal physiological conditions.

An example is the use of a prostate tumor array to determine that IGFBP2(Insulin Growth factor binding protein 2) is a marker associated withprogression of human prostate cancer. To elucidate mechanisms underlyingthe development and progression of hormone refractory prostate cancer,gene expression profiles were compared for four independent CWR22Rhormone refractory xenografts to androgen dependent CWR22 primaryxenograft. The CWR22 xenograft model of human prostate cancer wasestablished by transplantation of human prostate tumor cells into thenude mouse (Pretlow, J. Natl. Cancer Inst., 3:394-398, 1993). Thisparental tumor xenograft is characterized by secretion of prostatespecific antigen (PSA) and with rapid reduction of tumor size inresponse to the hormone-withdrawal therapy. Approximately half of thetreated animals will develop recurrent tumors from a few weeks toseveral months. These recurrent tumors are resistant to further hormonaltreatments when transferred to the new host. They also are characterizedby a more aggressive phenotype than parental CWR22 tumors, andeventually lead to death of the animal. This experimental model mimicsthe course of prostate cancer progression in human patients.

Comparison of the expression levels of 588 known genes during theprogression of the CWR22 prostate cancer in mice was performed with thecDNA microarray technology. RNA was prepared from CWR22 xenografts asdescribed earlier with minor modifications (Chirgwin, 1979). The mRNAwas purified using oligo(dT) selection with DynaBeads (Dynal) accordingto manufacturers instructions. The cDNA array hybridizations wereperformed on AtlasII cDNA arrays (Clontech) according to manufacturersinstructions. The cDNA probes were synthesized using 2 μg of polyA⁺ RNAand labeled with 32P a dCTP.

The gene expression pattern in a hormone-sensitive CWR22 xenograft wascompared with that of a hormone-refractory CWR22R xenograft.Expressional changes of several genes, which have previously been shownto be involved in prostate cancer pathogenesis were detected. Inaddition multiple genes were identified with no previous connection toprostate cancer, nor had they been known to be regulated by androgens.One of the most consistently upregulated genes, Insulin-like GrowthFactor Binding Protein 2 (IGFBP-2), was chosen for further study. Thetissue microarray technology was used to validate that the IGFBP2expression changes also take place in vivo, during the progression ofprostate cancer in patients undergoing hormonal therapy.

Formalin-fixed and paraffin-embedded samples from a total of 142prostate cancers were used for construction of the prostate cancertissue microarray. The tumors included 188 non-hormone refractoryprimary prostate cancers, 54 transurethral resection specimens oflocally recurrent hormone-refractory cancers operated during 1976-1997,and 27 transurethral resections for BPH as benign controls. The subsetof the primary non-hormone refractory tumors and benign controls wasselected from the archives of the Institute for Pathology, University ofBasel, (Switzerland), and the subset of hormone-refractory tumors fromthe University of Tampere (Finland). The group of primary non-hormonerefractory prostate cancers consisted of 50 incidentally detected tumorsin transurethral resections for presumed BPH (pT1a/b), and 138 radicalprostatectomy specimens of patients with clinically localized disease.The specimens were fixed in 4 percent phosphate-buffered formalin. Thesections were processed into paraffin and slides were cut at 5 μm andstained with haematoxylin and eosin (H & E). All sections were reviewedby one pathologist, and the most representative (usually the leastdifferentiated) tumor area was delineated on the slide. The tissuemicroarray technology was used as previously described to construct thetissue array.

Standard indirect immunoperoxidase procedures were used forimmunohistochemistry (ABC-Elite, Vector Laboratories). The goatpolyclonal antibody IGFBP-2, C-18 (1:x, Santa Cruz Biotechnology, Inc.,California) was used to detect IGFBP-2 after a microwave pretreatment.The reaction was visualized by diaminobenzidine as a chromogen. Positivecontrols for IGFBP-2 consisted of normal renal cortex. The primaryantibody was omitted for negative controls. The intensity of thecytoplasmic IGFBP-2 staining was estimated and stratified into 4 groups(negative, weak, intermediate, and strong staining).

There was a strong relationship between IGFBP-2 staining and progressionof cancer to a hormone refractory disease with an increasing frequencyof high-level staining. Strong IGFBP-2 staining was present in none ofthe normal glands, in 30% of the non-hormone-refractory primary tumorsbut in 96% of the recurrent, hormone-refractory prostate cancers(p=0.0001). Hence, this example provides another case in which ahigh-throughput expression survey by cDNA array hybridization indicateda specific gene, which may be involved in disease progression. Thishypothesis could be directly validated using the tissue arraytechnology. The results have identified IGFBP2 to be used as a targetfor developing diagnostic, prognostic or therapeutic approaches to themanagement of patients with advanced prostate cancer.

Example 12 PDGFB in Breast Cancer

The breast cancer SKBR3 cell line was screened with the AmpliOnc™ DNAarray, and Platelet Derived Growth Factor B (PDGFB) was identified asbeing amplified. Using this information, a PDGFB probe was made using aclone identical to the PDGFB clone used in the AmpliOnc™ array. Thisprobe was used to screen a breast cancer tumor array. It was found thatonly 2% of all the breast cancers screened were amplified for PDGFB. Amulti-tumor array (described in Example 6) was then probed using thisprobe. This revealed that, unexpectedly, the PDGFB gene was amplified ina large percentage of lung and bladder cancers. Thus, using theinvention, a novel marker of diagnostic importance in these other typesof tumors was identified.

Example 13 Herceptin Treatment

Tissue arrays can be used to screen large numbers of tumor tissuesamples to determine which tumors would be susceptible to a particulartreatment. For example, a breast cancer array can be screened forexpression of the HER-2 gene (also called ERBB2 in Example 1), asexplained in Example 1. Tumors that over-express and/or amplify theHER-2 gene may be good candidates for treatment with herceptin, which isan antibody that inhibits the expression of HER-2. Screening of themulti-tumor tissue array with the HER-2 antibodies or a DNA probe wouldprovide information about cancers other than breast cancer that could besuccessfully treated with the Herceptin therapy.

Example 14 Correlating Prognosis and Survival with Markers

Tumor tissue arrays constructed from tumors taken from patients for whomhistory and outcome is known may be used to assess markers withprognostic relevance. This example illustrates that prognostic markersin urinary bladder cancer can be evaluated using tumor tissue arrays, inspite of any intratumor heterogeneity.

An array of 315 bladder tumors was analyzed for nuclear p53 accumulationby immunohistochemistry. The p53 analysis was done twice; once onconventional large histological sections taken from entire tumor blocksand once on a section from a tumor array containing one sample from eachtumor. The tumor series consisted of 127 pTa, 81 pT1, and 128 pT2-4bladder carcinomas with clinical follow up information (tumor specificsurvival).

One block per tumor was analyzed. One section was taken from each blockfor immunohistochemical analysis. Then a tissue array was constructed bytaking one “punch biopsy” from each block and bringing it in an emptyrecipient block. Sections 4 μm thick were taken from primary tumorblocks and from the array block. The monoclonal-antibody DO-7 (DAKO,1:1000) was applied for immunostaining using standard procedures.

On large sections, a tumor was considered positive if moderate or strongnuclear p53 staining was seen in at least 20% of tumor cells, at leastin an area of the tumor. On array sections, a tumor was consideredpositive if moderate or strong nuclear p53 staining was seen in at least20% of arrayed tumor cells. Weak nuclear and any cytoplasmic p53staining was disregarded.

A Chi-square test was used to compare the p53 results between array andlarge sections. Survival curves were plotted according to Kaplan-Meier.A log rank test was applied to examine the relationship between p53positivity and tumor specific survival. Surviving patients were censoredat the time of their last clinical control. Patients dying from causesother than their bladder tumor were censored at the time of death.

Results showed that p53 could be analyzed on 315 arrayed tumor samples(21 samples were absent on the p53 stained array section). Onconventional sections, p53 immunostaining was positive in 105 of these315 tumors which were also present on the array. p53 positivity asdetected on conventional “large” sections was significantly linked topoor prognosis (FIG. 1A, p<0.0001). Only 69 of these 105 tumors (66%)that were p53 positive on large sections were also positive on arrayedtumor samples, while 36 (34%) remained negative probably because oftumor heterogeneity. Nevertheless, there was a strong associationbetween p53 immunostaining results on arrays and on large sections(p<0.0001) and p53 positivity on arrays was still significantly linkedto poor prognosis (FIG. 1B, p=0.0064).

The specific number of biopsies from each tumor that are preferablyobtained to reproduce 90%, 95%, or 100% of the information obtained fromthe whole-section analysis will make it possible to determine how many“punches” with the tissue arrays are required to extract clinicallysignificant information from the tissue array experiments. This optimalnumber may vary depending on the tumor type and the specific biologicaltarget that will be analyzed.

Example 15 Novel Gene Targets

Tissue arrays may be used to find novel targets for cancer and othertherapies. Hundreds of different genes may be differentially regulatedin a given cancer (based on cDNA, e.g., microarray, hybridizations, orother high-throughput expression screening methods such as sequencing orSAGE). Analysis of each gene candidate on a large tissue array can helpdetermine which is the most promising target for development of noveldrugs, inhibitors, etc. For instance, a tumor array containing thousandsof diverse tumor samples may be screened with a probe for an oncogene,or a gene coding for a novel signal transduction molecule. Such a probecan bind to one or a number of different tumor types. If a probe revealsthat a particular gene is overexpressed and/or amplified in many tumors,then that gene may be an important target, playing a key role in manytumors of one histological type or in different tumor types. Therapiesdirected to interfere with the expression of that gene, or with thefunction of the gene product of that gene, may be promising novel cancerdrugs. In particular, the tissue arrays can be used to help prioritizethe selection of targets for drug development.

Example 16 Tissue Array Followed by DNA Array

Although many of the foregoing examples have described the DNA arraybeing used prior to the tissue array, the present invention includes useof these arrays in either order, or in combination with other analytictechniques. Hence, genes of interest noted when probing multiple tumorsamples with a single probe during tissue array analysis cansubsequently be selected to be placed on a DNA array, using a uniquesequence from the gene of interest as one of the probes attached to thearray substrate. For example, one could tailor a DNA chip that has mostdiagnostic, prognostic, or therapeutic relevance based on informationfrom the microarray experiment.

Some possible interrelationship of cDNA arrays, CGH arrays, and tissuearrays is shown in FIG. 17. As illustrated in that figure, the variousassays can be performed in any order, or in any combination.

Example 17 Cell Line Arrays

Cultured cells or cells isolated from non-solid tissues or tumors (suchas blood samples, bone marrow biopsies, or cytological specimensobtained by needle aspiration biopsies) can also be analyzed with thetissue array techniques. This is an important extension of the tissuearray technology to the analysis of individual cells, or populations ofcells, obtained either directly from people or animals or after variousincubations of cell culture experiments have been performed in vitro(such as a specific hormonal or chemotherapeutic test performed on amicrotiter tray format for pharmaceutical drug screening). In theanalysis of malignancies, this would enable analysis of leukemias andlymphoma tissues or other liquid tumor types following the samestrategies described above for solid tumors.

Using this approach, cancer cell lines obtained from the American TypeCulture Collection (Rockville, Md.) were used. Cells were trypsinizedand the cell suspensions were spun down with a centrifuge at 1200×g. Thecell pellet was fixed with alcohol-based and formaldehyde fixatives, andthe fixed cell pellet was embedded in paraffin following routineprotocols used in pathology laboratories. The fixed and embedded cellsuspensions can then be used as starting material for the development ofcell arrays, using the same procedure as described previously for thefixed and embedded tissue specimens. It is anticipated that up to or atleast 1000 different cell populations can be arrayed in a singlestandard-size paraffin block using this method.

Very small punch sizes (for example less than 0.5 mm) can be used forcreating arrays from homogenous cultured cells. This allows high densityarrays to be constructed. For example, approximately 2000 different cellpopulations can be placed in a single 40 mm×25 mm paraffin block.

The methods of analyzing tissue in accordance with the present inventioncan take many different forms, other than those specifically disclosedin the above examples. The tissue specimens need not be abnormal, butcan be normal tissue analyzed for function and tissue distribution of aspecific gene, protein, or other biomarker (where a biomarker is abiological characteristic that is informative about a biologicalproperty of the specimen). The normal tissue could include embryonaltissues, or tissues from a genetically modified organism, such as atransgenic mouse.

The array technologies can also be used to analyze diseases that do nothave a genetic basis. For example, the gene or protein expressionpatterns that are likely to have importance for the pathogenesis ordiagnosis of a disease can be profiled. The tissue specimens need not belimited to solid tumors, but can also be taken from cell lines,hematological or other liquid tumors, cytological specimens, or isolatedcells.

Cells of humans or other animals can be used in a suspension, as maycells of yeast or bacteria. Alternatively, cells in suspension can bespun down in a centrifuge to provide a solid or semi-solid pellet,fixed, and then placed in the array, much like a tissue specimen. Liquidcellular suspensions can be placed with a pipette into a matrix (forexample depressions in a slide surface) and then can be analyzed in thesame manner as the tissue array already described. The tissue arrays canalso be used in cell line experiments, such as high throughputchemotherapeutic screening of cells grown on microtiter plates. Thecells from each well are treated with a different drug or a differentconcentration of the drug, and are then recovered and inserted into acell line microarray to analyze their functional characteristics,morphology, viability and expression of specific genes brought about bythe drug treatment.

Histological or immunological analyses that can be used with the arrayinclude, without limitation, a nucleic acid hybridization, PCR (such asin situ PCR), PRINS, ligase chain reaction, pad lock probe detection,histochemical in situ enzymatic detection, and the use of molecularbeacons. The tissue array technology can be used to directly collectspecimens (tissues or cells) from humans, animals, cell lines, or otherexperimental systems. For example, when biopsy specimens are treated ina conventional manner in pathology laboratories, after fixation, thespecimens are routinely inserted horizontally in a paraffin block.Therefore, it is very difficult, if not impossible to acquire specimensfrom such tissues into a tissue array. However, if multiple biopsyspecimens obtained from surgery are directly fixed (and, if required,embedded in a suitable medium, such as paraffin) and then inserteddirectly vertically into a matrix, this would enable construction of atissue array of biopsy specimens. Such an array would be useful forresearch purposes or in a clinical setting to e.g. monitor progressionof premalignant lesions or monitor treatment responses (with molecularmarkers) from metastatic tumors that cannot be surgically removed.

Cytological specimens (such as fine needle aspirations, cervicalcytology, blood specimens, isolated blood cells, or urine cells) can bepelleted by centrifugation and then fixed and embedded for arraying asexplained previously. Alternatively, cells can be fixed in a suspension,and directly inserted (e.g., pipetted) into holes in a matrix orembedded first, and then arrayed. This will provide an array of cellsfor research or for diagnostic purposes. This would enable rapidcytological diagnostics where multiple specimens from different patientscan be screened simultaneously from a single slide, not only for theirmorphology, but for their molecular characteristics. This would alsoenable automation of the analysis, since a number of specimens can bescreened with a microscope, automated image analysis system, scanner orassociated expert systems at once. The use of such cellular preparationsis particularly important for the diagnosis of hematological disorders,such as leukemias and lymphomas. This would also allow automation oflymphocyte typing from many patients at once, whose specimens areinserted in an array format for immunophenotyping or for analysis by insitu hybridization. Screening of donated blood specimens for viralantigens, viral DNA, or other pathogens in a blood bank could similarlybe performed in an array format.

Arrays of tumor progression can also be constructed by collectingspecimens from a subject at different stages of progression of thesubject's tumor (such as progression to hormone refractory prostatecancer). Alternatively, tumors of different stages from differentsubjects can be collected and incorporated into the array. The array canalso be used to follow the progression of pre-neoplastic lesions (suchas the evolution of cervical neoplasia), and the effects ofchemoprevention agents (such as the effects of anti-estrogens on breastepithelium and breast cancer development).

In another embodiment, specimens from a transgenic or model organism canbe obtained at different stages of development of the organism, such asdifferent embryonic stages, or different ages after birth. This enablesthe study of things such as normal and abnormal embryonic development.

The biological analyses that are performed on the microarray sectionscan be any analysis performed on regular tissue sections. Arrays canalso be assembled from one or more tumors at different stages ofprogression, such as normal tissue, hyperplasia, in situ cancer,invasive cancer, recurrent tumor, local lymph node metastases, ordistant metastases.

An “EST” or “Expressed Sequence Tag” refers to a partial DNA or cDNAsequence, typically of between 50 and 500 sequential nucleotides,obtained from a genomic of cDNA library, prepared from a selected cell,cell type, tissue or tissue type, organ or organism, which correspondsto an mRNA of a gene found in that library. An EST is generally a DNAmolecule.

“Specific hybridization” refers to the binding, duplexing, orhybridizing of a molecule only to a particular nucleotide sequence understringent conditions when that sequence is present in a complex mixture(e.g., total cellular) DNA or RNA.

OTHER EMBODIMENTS

In view of the many possible embodiments to which the principles of theinvention can be applied, it should be recognized that the illustratedembodiments are examples of the invention, and should not be taken as alimitation on the scope of the invention. Rather, the scope of theinvention is defined by the following claims. We therefore claim as ourinvention all that comes within the scope and spirit of these claims.

1. A method of detecting and distinguishing the presence ofhormone-refractory or metastatic prostate cell growth in a humanprostate tissue specimen, the method comprising: a. obtaining a prostatetissue specimen; b. providing a probe set comprising a labeled 8q24locus specific probe and an Xq12 locus specific probe; c. contactingsaid specimen with said labeled probes of step b), and; d. detecting thepresence of hormone-refractory prostate cancer cell growth or metastaticprostate cancer cell growth wherein Xq12 amplification in the humanprostate tissue sample greater than 8q24 amplification, if any, isindicative of hormone-refractory cell growth and, equivalent Xq12amplification and 8q24 amplification in the human prostate tissue sampleis indicative of metastatic prostate cancer cell growth.
 2. The methodof claim 1, wherein polymerase chain reaction (PCR) is used to determineif amplification is present.
 3. The method of claim 1, whereinfluorescence in situ hybridization (FISH) is used to determine ifamplification is present.
 4. The method of claim 1, wherein a cDNAmicroarray is used to determine if amplification is present.
 5. Themethod of claim 1, wherein in step b) a labeled X centromeric probe anda labeled chromosome 8 centromeric probe are also provided and 8q24and/or Xq12 are considered amplified if said loci are amplified at least3 times more than the signals for the respective centromeric signals percell in at least 10% of identified tumor cells.